Methods to determine KDM1A target engagement and chemoprobes useful therefor

ABSTRACT

The invention relates to methods to determine KDM1A target engagement and chemoprobes useful therefor. In particular, the invention relates to non-peptidic KDM1A chemoprobes carrying a tag or label that can be used to assess KDM1A target engagement in cells and tissues. These chemoprobes can also be used to identify KDM1A interacting factors and analyze expression levels of KDM1A.

This application is a national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/EP2017/056330, filed on Mar. 16, 2017,which claims priority of European Patent Application 16382119.2, filedMar. 16, 2016. The contents of these applications are each incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates to methods to determine KDM1A target engagementand chemoprobes useful therefor. In particular, the invention relates tonon-peptidic KDM1A chemoprobes carrying a tag or label that can be usedto assess KDM1A target engagement in cells and tissues. Thesechemoprobes can also be used to identify KDM1A interacting factors andanalyze expression levels of KDM1A.

BACKGROUND

Covalent modifications of histones are closely related to the control ofgene transcription. Chromatin modifications constitute an epigeneticcode that is dynamically ‘written’ and ‘erased’ by specialized proteins,and ‘read’ or interpreted by proteins that translate the code into geneexpression changes. Acetylation, phosphorylation and methylation ofhistones is mediated by histone acetyl (HATs), phospho- and methyltransferases (PRMTs, HMTs), and can be reversed by deacetylases (HDACs),phosphatases and demethylases (KDMs),

KDMs are classified in 2 families with distinct enzymatic mechanisms ofaction: FAD-dependent amine oxidases (KDM1), which demethylateH3K4me1/2, and JmjC domain-containing KDMs, which are Fe(II)-dependentenzymes that catalyze the demethylation of mono-, di- and trimethylatedlysines.

KDM1A (Lysine Specific Demethylase-1, also known as LSD1 or AOF2)belongs to the family of FAD-dependent amine oxidases and demethylateshistone lysines via a hydride transfer from the N6-methylgroup of themethylated lysine to the FAD cofactor, forming an unstable imineintermediate that is further hydrolyzed to liberate formaldehyde. Thiscatalytic mechanism permits demethylation of secondary and tertiary butnot of quaternary amines, limiting the substrate to mono anddimethylated lysines. During the demethylation reaction, the FADcofactor is reduced to FADH2 and subsequently reoxidized to FAD, aprocess in which H₂O₂ is released. KDM1A is structurally related to thefamily of the monoamine oxidases, which include MAO-A, MAO-B, SMOX andILI1.

KDM1A is a key enzyme involved in various biological processes includinghematopoiesis, embryonic development, neurodevelopment, and viralbiology. Increased or inappropriate KDM1A expression has been describedin leukemogenesis and in solid tumors. KDM1A has been recognized as aninteresting target for the development of new drugs to treat cancer,neurological diseases and other conditions, and a number of KDM1Ainhibitors are currently under preclinical or clinical development foruse in human therapy.

Assessment of KDM1A target engagement by KDM1A inhibitors in cells ortissues has been analyzed most frequently indirectly, throughmeasurement of me2H3K4 accumulation, or by assessment of changes in geneexpression. However, inhibition of histone lysine modifiers often leadsto local rather than global changes in histone marks, histonemodifications can be modulated by more than one cellular enzyme (forexample H3K4 can be methylated by the SET/MLL proteins, and themethylation can be reversed by the KDM1 and KDM5 demethylases), and itis not always clear if a given change in histone mark is a direct orindirect consequence of treatment.

A NanoBRET assay that assesses LSD1 detachment from H3 histones uponKDM1A inhibition has also been described (JP McGrath et al., Cancer Res2016, epub 2 Feb. 2016, PMID: 26837761). However, said assay requirestransfection of cells with modified KDM1A and H3 constructs, and cannotreadily be applied to samples obtained from animals or from humansubjects.

A reliable method for the quantitative assessment of inhibitorengagement to KDM1A in cells and tissues, amenable to the analysis ofclinical samples, is thus needed.

SUMMARY OF THE INVENTION

The invention relates to methods for the direct determination of KDM1Atarget engagement in a sample which is based on the determination offree KDM1A using specific KDM1A chemoprobes as described herein and inthe appended claims. The methods as described herein can be used toquantify KDM1A target engagement of a KDM1A inhibitor in all kinds ofsamples, including clinical samples. The invention also relates to saidKDM1A chemoprobes and to further uses thereof, as described herein andin the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: KDM1A Western blot of pull down of cellular protein extractsdescribed in example 3 using the chemoprobe example 1.6. (FIG. 1A) andof cell protein extracts treated with ORY-1001 (FIG. 1B) according toconditions detailed in example 3.

FIG. 2: Western blot using KDM1A or SMOX antibody, the pull-down productwas mediated by chemoprobe example 1.6 (described in example 4)

FIG. 3: Scheme of sandwich ELISA for determination of total KDM1A (FIG.3A) and KDM1A chemoprobe capture ELISA based determination of free KDM1A(FIG. 3B) as described in Example 5

FIG. 4: Total and free rKDM1A calibration curves as described in example5.

FIG. 5: Total (FIG. 5A) and free (FIG. 5B) KDM1A in human PBMCs fromhealthy donors according to example 5.

FIG. 6: Detection of free (FIG. 6A) and total (FIG. 6B) KDM1A byAlphaLISA as explained in example 6.

FIG. 6C: detection of free KDM1A by AlphaLISA in the presence of 5 uMORY-1001 as described in Example 6.

FIG. 7: IC50 determination of KDM1A target engagement in cells (MV4;11)treated with ORY-1001 and using the luminescent chemoprobe immunoassaydescribed in example 7

FIG. 8: KDM1A Target engagement in cells after treatment with differentKDM1A inhibitors detailed in example 7. FIG. 8A: KDM1A Target engagementin MV(4;11) leukemic cells; FIG. 8B: KDM1A Target engagement in THP-1leukemic cells; FIG. 8C: KDM1A Target engagement in LNCap prostatecancer cells.

FIG. 9: KDM1A Target engagement in rat PBMCs (FIG. 9A) or rat lung (FIG.9B) treated with ORY-1001 at different doses according to the conditionsdescribed in example 8. FIG. 9C: KDM1A Target engagement in brain ofSAMP8 mice treated with Compound C as described in example 8.

FIG. 10: Determination of dynamics of KDM1A target engagement in ratpooled PBMCs treated with ORY-1001 according to the conditions detailedin example 9.

FIG. 11: Determination of KDM1A target engagement in PBMCs of humanhealthy volunteers treated with Compound C as described in example 9.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the specification and appended claims, unless specified tothe contrary, the following terms have the meaning indicated below.

Sample: any tissue, cells, protein extract, recombinant protein to whichthe methods of the invention described herein is to be applied. A sampleor the subject from which the sample has been obtained may have beentreated with a KDM1A inhibitor.

Reference sample: any tissue, cells, protein extract, recombinantprotein that is chosen to serve as a reference to the sample. Areference sample or the subject from which the reference sample has beenobtained may have been treated with a vehicle, may have been obtainedprior to treatment with a KDM1A inhibitor, or may have been obtainedwhen the effect of the KDM1 A inhibitor has ceased completely.

Subject: human or animal organism.

in vitro: outside the context of a living organism; i.e. not in vivo.

Free KDM1A: enzymatically active KDM1A enzyme present in a sample, i.e.KDM1A not bound by a KDM1A inhibitor. Free KDM1A is available forbinding to a KDM1A chemoprobe.

Total KDM1A: all KDM1A enzyme present in a sample, whether free,inhibited by a KDM1A inhibitor, or bound by a KDM1A chemoprobe.

Level: the amount of signal of free KDM1A or total KDM1A in a sample orsubject determined by the methods described herein. The amount of signalcan also be obtained from raw signal readouts by data processing. Dataprocessing may comprise operations to remove background signal,eliminate outliers, average and/or normalize the raw signal usingmethods described in the literature. The level can also refer to theconcentration/absolute amount of free KDM1A or total KDM1A in a sample,calculated by interpolating the signal of free KDM1A or total KDM1A in acalibration curve generated using a dilution series of samples withknown free KDM1A or total KDM1A concentrations/amounts.

KDM1A inhibitor: herein is a non-peptidic compound that inhibits theenzymatic activity of KDM1A. The term KDM1A inhibitor will be usedherein exclusively for a compound that is not a KDM1A chemoprobeaccording to the invention.

Non-peptidic compound: a compound that does not contain a chain of atleast 3 amino acid monomers linked by peptide (amide) bonds. Thepresence of an (single) amide bond will not in itself be sufficient toterm a compound as peptidic.

KDM1A chemoprobe (also termed chemoprobe): compound that selectivelybinds and inhibits KDM1A and incorporates a label or tag for detection,particularly a compound of formula I, II, IIa, III, IIIa as describedherein and in the appended claims. The term KDM1A chemoprobe will beused herein exclusively for a compound that is not a KDM1A inhibitoraccording to the invention.

Tag: herein tag will be used to describe a moiety that can beincorporated into a molecule, for example into a KDM1A chemoprobe, thatcan be recognized and used in capture, recovery, isolation, purificationor detection procedure of that molecule or of complexes involving thatmolecule.

Label: herein label will be used to describe a moiety that can beincorporated into a molecule, for example into a KDM1A chemoprobe ordetection agent, and that directly gives rise to a signal, for example afluorescent, bioluminescent, isotope, mass spectrometry label. A labelcan also be used as a tag if a suitable capture agents is available, forexample a fluorescein moiety can be recognized and captured by ananti-fluorescein antibody.

Capture agent: agent that recognizes and binds the tag and that can beused to capture the chemoprobe or chemoprobe complexes from their crudebiological source. In this invention, the capture agent can also be anagent that can be used to capture KDM1A or KDM1A containing complexesfrom their crude biological source.

Detection agent: agent that recognizes and binds a tag and that can beused to detect the KDM1A chemoprobe or KDM1A chemoprobe bound complexes,or an agent that can be used to detect KDM1A or a KDM1A containingcomplex in a sample.

Irreversible KDM1A inhibitor: a KDM1A inhibitor that reacts with KDM1Aor its cofactor and changes it chemically (e.g. via covalent bondformation), irreversibly inactivating the KDM1A enzyme activity.Examples of irreversible KDM1A inhibitors include FAD binding KDM1Ainhibitors.

Reversible KDM1A inhibitor: a KDM1A inhibitor that binds non-covalentlyto KDM1A or its cofactor and reversibly inactivates the KDM1A enzymeactivity.

Target engagement: the target engagement of a KDM1A inhibitor is ameasure for the degree of occupation of KDM1A by a KDM1A inhibitor.

Pharmacodynamics: herein, the study of the time dependent relation ofthe dose of KDM1A inhibitor administered, and the KDM1A targetengagement.

Antibody as used herein is intended to include monoclonal antibodies,polyclonal antibodies, and chimeric antibodies, as well as Fabfragments, ScFv and the like.

KDM1A Chemoprobes

In one aspect, the invention relates to compounds that can be used asKDM1A chemoprobes, for use in the methods as described herein.

Accordingly, the invention provides a compound of formula (I)P-L-Z  (I)

or a salt thereof,

wherein:

P is a tag or label;

L is a divalent C₆₋₁₀₀ hydrocarbon group, wherein one or more carbonatoms comprised in said hydrocarbon group are each optionally replacedby a heteroatom selected independently from O, S and N, wherein one ormore carbon atoms comprised in said hydrocarbon group are eachoptionally replaced by a group selected independently from the groupconsisting of —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹— and —NR¹—SO₂—, and

wherein L provides a distance of at least 6 atoms between P and Z;

R¹ is hydrogen or C₁₋₄ alkyl; and

Z is a radical of a KDM1A inhibitor.

As used herein in connection with Z, a radical of a KDM1A inhibitor ismeant to be a KDM1A inhibitor where a H atom is replaced by theconnecting bond.

In the compounds of formula (I) the KDM1A inhibitor can be anirreversible KDM1A inhibitor or a reversible KDM1A inhibitor.Preferably, the KDM1A inhibitor is an irreversible KDM1A inhibitor andthus Z preferably is a radical of an irreversible KDM1A inhibitor.

KDM1A inhibitors for use in the context of a compound of formula (I)include any of the compounds disclosed under the section “KDM1Ainhibitors” below.

Preferably, the KDM1A inhibitor is a selective KDM1A inhibitor, e.g. aselective irreversible KDM1A inhibitor. As used herein, a “selectiveKDM1A inhibitor” refers to a KDM1A inhibitor which exhibits aselectivity of at least 10-fold for KDM1A over other FAD-dependentmonoamine oxidases, particularly MAO-A and MAO-B. More preferably, thecompound exhibits a selectivity of at least 30-fold for KDM1A over otherFAD-dependent monoamine oxidases, particularly MAO-A and MAO-B, andstill more preferably of at least 50-fold for KDM1A over otherFAD-dependent monoamine oxidases, particularly MAO-A and MAO-B. Theability of a compound to inhibit KDM1A and other FAD-dependent monoamineoxidases, particularly MAO-A and MAO-B are preferably to be determinedin accordance with the assays described in the appended Examples.

In some embodiments, the KDM1A inhibitor in a compound of formula (I) isnot parnate, also known as tranylcypromine, which is not a selectiveKDM1A inhibitor.

In the compounds of formula (I) the KDM1A inhibitor is preferably anirreversible KDM1A inhibitor comprising a 2-cyclyl-cyclopropylaminomoiety, preferably a 2-(hetero)arylcyclopropylamino compound, as definedherein.

In certain embodiments, in the compounds of formula (I) Z is a radicalof an irreversible KDM1A inhibitor disclosed in WO2010/043721,WO2010/084160, WO2011/035941, WO2011/042217, WO2011/131697,WO2012/013727, WO2012/013728, WO2012/045883, WO2013/057320,WO2013/057322, WO2012/135113, WO2013/022047, WO2014/058071,WO2010/143582, US2010/0324147, WO2011/131576, WO2014/084298,WO2014/086790, WO2014/164867, WO2015/021128, WO2015/123408,WO2015/123424, WO2015/123437, WO2015/123465, WO2015/156417 orWO2015/181380. In certain embodiments, in the compounds of formula (I) Zis a radical of an irreversible KDM1A inhibitor disclosed inWO2010/043721, WO2010/084160, WO2011/035941, WO2011/042217,WO2011/131697, WO2012/013727, WO2012/013728, WO2012/045883,WO2013/057320, WO2013/057322, WO2012/135113, WO2013/022047,WO2014/058071, WO2010/143582, US2010/0324147, WO2011/131576,WO2014/084298, WO2014/086790, WO2014/164867, WO2015/021128,WO2015/123408, WO2015/123424, WO2015/123437, WO2015/123465,WO2015/156417, WO2015/181380, WO2016/123387, WO2016/130952,WO2016/172496, WO2016/177656, WO2017/027678 or CN106045862.

In certain embodiments, the KDM1A inhibitor is preferably a compound offormula (A), (B), (C), (D), (E), (F), (G), (H), (J), (K), (L), (M) or(N), as described in more detail below. More preferably, the KDM1Ainhibitor is a compound of formula (C), (F), (H), (J), (K), (L), (M) or(N). Still more preferably, the KDM1A inhibitor is a compound from thelists of examples provided below for compounds of formulae (C), (F),(H), (J), (K) or (L).

Preferably, in the compounds of the invention described herein the KDM1Ainhibitor is(trans)-N1-((1R,2S)-2-phenylcyclopropyl)cyclohexane-1,4-diamine (alsoknown as ORY-1001 or RG6016) or a salt thereof.

The invention also relates to a compound of formula (I) wherein Z is agroup of formula Z1

wherein:

A is aryl or heteroaryl, wherein said aryl or said heteroaryl isoptionally substituted;

B is aryl, heteroaryl or heterocycloalkyl, wherein B is optionallysubstituted;

m is 0 or 1;

Y is a bond, —(C₁₋₄ alkylene)-O—, —(C₁₋₄ alkylene)-NR²—, —(C₀₋₄alkylene)-C(═O)—NR²—, or —(C₀₋₄ alkylene)-NR²—C(═O)—;

R² is hydrogen or C₁₋₄ alkyl;

D is hydrogen, —(C₁₋₄ alkylene)-CO—NR³R⁴, cyclyl or —(C₁₋₄alkylene)-cyclyl, wherein cyclyl and the cyclyl moiety in the —(C₁₋₄alkylene)-cyclyl are each optionally substituted;

R³ and R⁴ are each independently selected from hydrogen, C₁₋₄ alkyl and—(C₀₋₄ alkylene)-cyclyl, wherein the cyclyl moiety in the —(C₀₋₄alkylene)-cyclyl is optionally substituted, or R³ and R⁴ are linkedtogether to form together with the N atom to which they are bound aheterocyclic ring which may contain one or more additional heteroatomsselected from N, O and S and which is optionally substituted;

and the groups —(B—Y)_(m)-A- and —NH-D on the cyclopropyl ring are intrans configuration.

In any chemical drawings depicted herein, the waved line (interruptedbond) refers to the attachment point of the group (Z1 in this case, orany other group, as applicable) to the remainder of the compound (i.e.to L in the case of Z1).

In Z1, D is hydrogen, —(C₁₋₄ alkylene)-CO—NR³R⁴, cyclyl or —(C₁₋₄alkylene)-cyclyl, wherein cyclyl and the cyclyl moiety in the —(C₁₋₄alkylene)-cyclyl are each optionally substituted.

Preferably, D is —(C₁₋₄ alkylene)-CO—NR³R⁴, cyclyl or —(C₁₋₄alkylene)-cyclyl, wherein cyclyl and the cyclyl moiety in the —(C₁₋₄alkylene)-cyclyl are each optionally substituted.

More preferably, D is cyclyl or —(C₁₋₄ alkylene)-cyclyl, wherein cyclyland the cyclyl moiety in the —(C₁₋₄ alkylene)-cyclyl are each optionallysubstituted.

Still more preferably, D is cycloalkyl, benzocycloalkyl,heterocycloalkyl or —(C₁₋₄ alkylene)-cyclyl, wherein the cycloalkyl, thebenzocycloalkyl, the heterocycloalkyl and the cyclyl moiety in the—(C₁₋₄ alkylene)-cyclyl are each optionally substituted.

In some embodiments, D is optionally substituted cycloalkyl oroptionally substituted benzocycloalkyl, preferably optionallysubstituted cycloalkyl. Even more preferably, D is a group of formula

In some embodiments, D is optionally substituted heterocycloalkyl, forexample D is optionally substituted piperidinyl, preferably optionallysubstituted 4-piperidinyl.

In some embodiments, D is —(C₁₋₄ alkylene)-cyclyl wherein the cyclylmoiety in the —(C₁₋₄ alkylene)-cyclyl is optionally substituted.

In some embodiments, D is —(C₁₋₁₄alkylene)-cycloalkyl wherein thecycloalkyl in the —(C₁₋₄ alkylene)-cycloalkyl is optionally substituted.In some embodiments, D is cyclopropylmethyl.

In some embodiments, D is —(C₁₋₄ alkylene)-heterocycloalkyl, wherein theheterocycloalkyl in the —(C₁₋₄ alkylene)-heterocycloalkyl is optionallysubstituted. In some embodiments, D is —CH₂-heterocycloalkyl, morepreferably —CH₂-(4-piperidinyl), wherein the heterocycloalkyl in the—CH₂-heterocycloalkyl and the 4-piperidinyl in the —CH₂-(4-piperidinyl)are each optionally substituted. In some embodiments, D is a group offormula

In some embodiments, D is a group of formula

In some embodiments, D is —(C₁₋₄ alkylene)-heteroaryl, preferably—CH₂-heteroaryl, wherein the heteroaryl in the —(C₁₋₄alkylene)-heteroaryl and the heteroaryl in the —CH₂-heteroaryl isoptionally substituted. Preferably, the heteroaryl in D is a monocyclic5- or 6-membered heteroaryl, which is optionally substituted. In someembodiments, D is a group of formula

In Z1, Y is a bond, —(C₁₋₄ alkylene)-O—, —(C₁₋₄ alkylene)-NR²—, —(C₀₋₄alkylene)-C(═O)—NR²—, or —(C₀₋₄ alkylene)-NR²—C(═O)—. Although bothorientations are possible for Y groups, preferably Y groups are linkedto A through the O, NR2 or C(═O) groups, respectively, and are linked toB through the alkylene groups.

Preferably Y is a bond, —CH₂—O—, —C(═O)—NR²—, or —NR²—C(═O)—. Morepreferably, Y is a bond.

In Z1, A is aryl or heteroaryl, wherein said aryl or said heteroaryl isoptionally substituted. In relation to A, aryl and heteroaryl relate toan aryl or heteroaryl group as defined below. In addition, in relationto A, aryl and heteroaryl also encompasses ring systems comprising atleast an aromatic ring. Thus rings comprising an aromatic ring likephenyl or an heteroaromatic ring like pyridyl fused to a non-aromaticcarbocyclic or heterocyclic ring are also included, for example2,3-dihydrobenzofuran. Preferably, the two points of attachment of ringA to the remainder of the molecule are on the aromatic ring.

Preferably, A is optionally substituted phenyl. In some embodiments, Ais unsubstituted phenyl.

In Z1, B is aryl, heteroaryl or heterocycloalkyl, wherein B isoptionally substituted. Preferably, B is aryl or heteroaryl, wherein Bis optionally substituted. More preferably, B is optionally substitutedaryl, preferably optionally substituted phenyl. In some embodiments, Bis unsubstituted phenyl.

In some embodiments, m is 1 and Y is bond, more preferably m is 1, Y isbond, and B is optionally substituted aryl, preferably optionallysubstituted phenyl. In some embodiments, B is unsubstituted phenyl.

In some embodiments, m is 0.

The invention further provides a compound of formula (I) as definedabove, wherein Z is a group of formula Z2

wherein the phenyl ring is optionally substituted, and wherein thesubstituents on the cyclopropyl ring are in trans configuration. In someembodiments, the phenyl ring is unsubstituted.

The invention further provides a compound of formula (I) as definedabove, wherein Z is a group of formula Z3a or Z3b

wherein in Z3a and Z3b the phenyl rings are optionally substituted andthe substituents on the cyclopropyl ring are in trans configuration. Insome embodiments, the phenyl rings are unsubstituted.

The invention further provides a compound of formula (I) as definedabove, wherein Z is a group of formula Z4:

wherein the phenyl ring is optionally substituted. Preferably, thephenyl ring is unsubstituted.

The invention further provides a compound of formula (I) as definedabove, wherein Z is a group of formula Z5a or Z5b:

wherein in Z5a and Z5b the phenyl rings are optionally substituted.Preferably, the phenyl rings are unsubstituted.

In a compound of formula (I), P is a tag or label. Examples of tags orlabels that can be used in a compound of formula (I) include the tagsand labels described in more detail in the corresponding section, below.

In a compound of formula (I), L is a divalent C₆₋₁₀₀ hydrocarbon group,wherein one or more carbon atoms comprised in said hydrocarbon group areeach optionally replaced by a heteroatom selected independently from O,S and N, wherein one or more carbon atoms comprised in said hydrocarbongroup are each optionally replaced by a group selected independentlyfrom the group consisting of —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—,—NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—,—C(═O)—O—, —O—C(═O)—, —SO₂—NR¹— and —NR¹—SO₂—, and wherein L provides adistance of at least 6 atoms between P and Z.

Preferably, in the compounds of formula (I) L is a divalent C₃₀-C₈₀hydrocarbon group, wherein one or more carbon atoms comprised in saidhydrocarbon group are each optionally replaced by a heteroatom selectedindependently from O, S and N, wherein one or more carbon atomscomprised in said hydrocarbon group are each optionally replaced by agroup selected independently from the group consisting of —C(═O)—,—NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,—O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—, —SO₂—NR¹— and—NR¹—SO₂—, and wherein L provides a distance between P and Z of 25 to 70atoms.

More preferably, L is a divalent C₄₀-C₇₀ hydrocarbon group, wherein oneor more carbon atoms comprised in said hydrocarbon group are eachoptionally replaced by a heteroatom selected independently from O, S andN, wherein one or more carbon atoms comprised in said hydrocarbon groupare each optionally replaced by a group selected independently from thegroup consisting of —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—,—NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—,—C(═O)—O—, —O—C(═O)—, —SO₂—NR¹— and —NR¹—SO₂—, and wherein L provides adistance between P and Z of 35 to 65 atoms.

In some embodiments, L comprises a heteroalkylene group of 6 to 70atoms, preferably of 6 to 50 atoms.

Preferably, in a compound of formula (I) L comprises a group of formula(i) or (ii):

(i) —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is an integer from 6 to 20,preferably from 8 to 18, and p is an integer from 1 to 5; or

(ii) —(CH₂CH₂O)_(x)—(CH₂)_(q)-G-(CH₂CH₂O)_(y)(CH₂)_(r)—, wherein G is—NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,—O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, or —O—C(═O)—, preferably G is—NR¹—C(═O)— or —C(═O)—NR¹—, one of x and y is an integer from 1 to 18and the other from x and y is an integer from 0 to 17 provided that x+yis from 4 to 18, preferably x+y is from 6 to 16, and each of q and r isan integer independently selected from 1 to 5.

More preferably, in a compound of formula (I) L comprises a group offormula (i) or (ii):

(i) —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is an integer from 6 to 20,preferably from 8 to 18, and p is an integer from 1 to 5; or

(ii) —(CH₂CH₂O)_(x)—(CH₂)_(q)-G-(CH₂CH₂O)_(y)(CH₂)_(r)—, wherein G is—NR¹—C(═O)— or —C(═O)—NR¹—, one of x and y is an integer from 1 to 18and the other from x and y is an integer from 0 to 17 provided that x+yis from 4 to 18, preferably x+y is from 6 to 16, and each of q and r isan integer independently selected from 1 to 5.

Still more preferably, in a compound of formula (I) L comprises a groupof formula —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is an integer from 6 to20, preferably from 8 to 18, and p is an integer from 1 to 5.

Preferably, L comprises a group of formula X₁—X₂—X₃, wherein X₁ islinked to the remainder of L and X₃ is linked to Z, and wherein:

X₁ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹—, —NR¹—SO₂— or —O—;

X₂ is C₀₋₅ alkylene;

X₃ is arylene or heteroarylene, wherein said arylene and saidheteroarylene are each optionally substituted.

Preferably, X₁ is —NHC(═O)—, —C(═O)NH—, —SO₂—NH— or —NH—SO₂—, morepreferably X₁ is —NHC(═O)— or —C(═O)NH—, still more preferably—NHC(═O)—.

Preferably, X₃ is connected to X₁—X₂ and to Z in a 1,3 disposition. Morepreferably, X₃ is a group of formula:

wherein each ring is optionally substituted. Still more preferably,wherein X₃ is a group of formula:

which is optionally substituted. In some embodiments, X₃ isunsubstituted.

Preferably, X₂ is C₁₋₅ alkylene, more preferably —(CH₂)₁₋₅—.

In some embodiments, L comprises a group of formula X₁—X₂—X₃, wherein X₁is —NHC(═O)— or —C(═O)NH—, X₂ is C₁₋₅ alkylene and X₃ is a group offormula:

Preferably, L comprises a group of formula X₁—X₂—X₃, wherein X₁—X₂—X₃ isa group of formula:

wherein the group is linked to Z through the phenyl ring and to theremainder of L through the N atom.

Particularly preferred compounds for use as KDM1A chemoprobes accordingto the invention include the compounds of formula (II).

Accordingly, the invention provides a compound of formula (II)

or a salt thereof,

wherein:

P is a tag or label;

D is cyclyl or —(C₁₋₄ alkylene)-cyclyl, wherein cyclyl and the cyclylmoiety in the —(C₁₋₄ alkylene)-cyclyl are each optionally substituted;

L₁ is a divalent C₆₋₉₀ hydrocarbon group, wherein one or more carbonatoms comprised in said hydrocarbon group are each optionally replacedby a heteroatom selected independently from O, S and N, wherein one ormore carbon atoms comprised in said hydrocarbon group are eachoptionally replaced by a group selected independently from the groupconsisting of —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹— and —NR¹—SO₂—, and wherein L₁ provides a distance of at least3 atoms between P and X₁;

R¹ is hydrogen or C₁₋₄ alkyl;

X₁ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹—, —NR¹—SO₂— or —O—;

X₂ is C₀₋₅ alkylene;

one of Z₁ and Z₂ is CH or N, and the other of Z₁ and Z₂ is CH;

s and t are each independently selected from 0, 1 and 2;

R₅ and R₆ are at each occurrence independently selected from C₁₋₄ alkyl,halo, —NH₂, —NR^(a)R^(c), —CN, —OH, —OR^(c), haloC₁₋₄ alkyl, cyclyl,cyclylC₁₋₄alkyl-, and C₁₋₄alkyl-O—C₁₋₄alkyl;

R^(a) is selected from hydrogen, C₁₋₄alkyl and haloC₁₋₄alkyl;

R^(c) is independently selected from C₁₋₄ alkyl, haloC₁₋₄alkyl, cyclyl,cyclylC₁₋₄alkyl-, and C₁₋₄alkyl-O—C₁₋₄alkyl-;

and wherein the phenyl and —NH-D groups on the cyclopropyl ring are intrans configuration.

In another aspect, the invention provides a compound of formula (IIa)

or a salt thereof

wherein the meanings for the various substituents are as defined above.

Throughout this document, the symbols R and S on the cyclopropyl ringare meant to indicate absolute configuration on this ring.

Still more preferably, D is cycloalkyl, benzocycloalkyl,heterocycloalkyl or —(C₁₋₄ alkylene)-cyclyl, wherein the cycloalkyl, thebenzocycloalkyl, the heterocycloalkyl and the cyclyl moiety in the—(C₁₋₄ alkylene)-cyclyl are each optionally substituted.

In some embodiments, in a compound of formula (II) or (IIa), D isoptionally substituted cycloalkyl or optionally substitutedbenzocycloalkyl, preferably optionally substituted cycloalkyl. Even morepreferably, D is a group of formula

In some embodiments, in a compound of formula (II) or (IIa), D isoptionally substituted heterocycloalkyl, for example D is optionallysubstituted piperidinyl, preferably optionally substituted4-piperidinyl.

In some embodiments, in a compound of formula (II) or (IIa), D is —(C₁₋₄alkylene)-cyclyl wherein the cyclyl moiety in the —(C₁₋₄alkylene)-cyclyl is optionally substituted.

In some embodiments, in a compound of formula (II) or (IIa), D is —(C₁₋₄alkylene)-cycloalkyl wherein the cycloalkyl in the —(C₁₋₄alkylene)-cycloalkyl is optionally substituted. In some embodiments, Dis cyclopropylmethyl.

In some embodiments, in a compound of formula (II) or (IIa), D is —(C₁₋₄alkylene)-heterocycloalkyl, wherein the heterocycloalkyl in the —(C₁₋₄alkylene)-heterocycloalkyl is optionally substituted. In someembodiments, D is —CH₂-heterocycloalkyl, more preferably—CH₂-(4-piperidinyl), wherein the heterocycloalkyl in the —CH₂—heterocycloalkyl and the 4-piperidinyl in the —CH₂-(4-piperidinyl) areeach optionally substituted. In some embodiments, D is a group offormula

In some embodiments, D is a group of formula

In some embodiments, in a compound of formula (II) or (IIa), D is —(C₁₋₄alkylene)-heteroaryl, preferably —CH₂— heteroaryl, wherein theheteroaryl in the —(C₁₋₄ alkylene)-heteroaryl and the heteroaryl in the—CH₂-heteroaryl is optionally substituted. Preferably, the heteroaryl inD is a monocyclic 5- or 6-membered heteroaryl, which is optionallysubstituted. In some embodiments, D is a group of formula

In a further aspect, the invention provides a compound of formula (III),or a salt thereof

wherein the substituents on the cyclopropyl ring are in transconfiguration, wherein the meanings for the various substituents are asdefined above.

A particularly preferred compound is a compound of formula (IIIa), or asalt thereof

wherein the meanings for the various substituents are as defined above.

Preferably, in a compound of formula (II), (IIa), (III) or (IIIa), X₁ is—NR¹—C(═O)—, —C(═O)—NR¹—, —SO₂—NR¹— or —NR¹—SO₂—, more preferably X₁ is—NHC(═O)—, —C(═O)NH—, —SO₂—NH— or —NH—SO₂—, still more preferably X₁ is—NHC(═O)— or —C(═O)NH—, and even more preferably X₁ is —NHC(═O)—.

Preferably, in a compound of formula (II), (IIa), (III) or (IIIa), X₂ isC₁₋₅ alkylene, preferably —(CH₂)₁₋₅—.

Preferably, in a compound of formula (II), (IIa), (III) or (IIIa) X₁—X₂is a group of formula

wherein the group is linked to the ring through the carbon atom and toL₁ through the N atom.

Preferably, in a compound of formula (II), (IIa), (III) or (IIIa) eachof Z₁ and Z₂ is CH.

Preferably, in a compound of formula (II), (IIa), (III) or (IIIa), R₅and R6 are each independently selected from halo and C₁₋₄ alkyl.

In some embodiments, each of s and t is 0.

Preferably, in a compound of formula (II), (IIa), (III) or (IIIa), L₁ isa group of formula X₄—X₅, wherein X₄ is linked to P and X₅ is linked toX₁, wherein:

X₄ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹— or —NR¹—SO₂—; and

X₅ is a group of formula (i) or (ii):

(i) —(CH₂CH₂O)_(x)—(CH₂)_(p)—, wherein n is an integer from 6 to 20,preferably from 8 to 18, and p is an integer from 1 to 5; or

(ii) —(CH₂CH₂O)_(x)—(CH₂)_(q)-G-(CH₂CH₂O)_(y)(CH₂)_(r)—, wherein G is—NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,—O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, or —O—C(═O)—, preferably G is—NR¹—C(═O)— or —C(═O)—NR¹—, one of x and y is an integer from 1 to 18and the other from x and y is an integer from 0 to 17 provided that x+yis from 4 to 18, preferably x+y is from 6 to 16, and each of q and r isan integer independently selected from 1 to 5.

More preferably, in a compound of formula (II), (IIa), (III) or (IIIa),L₁ is a group of formula X₄—X₅, wherein X₄ is linked to P and X₅ islinked to X₁, wherein:

X₄ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹— or —NR¹—SO₂—; and

X₅ is a group of formula (i) or (ii):

(i) —(CH₂CH₂O)_(x)—(CH₂)_(p)—, wherein n is an integer from 6 to 20,preferably from 8 to 18, and p is an integer from 1 to 5; or

(ii) —(CH₂CH₂O)_(x)—(CH₂)_(q)-G-(CH₂CH₂O)_(y)(CH₂)_(r)—, wherein G is—NR¹—C(═O)— or —C(═O)—NR¹—, one of x and y is an integer from 1 to 18and the other from x and y is an integer from 0 to 17 provided that x+yis from 4 to 18, preferably x+y is from 6 to 16, and each of q and r isan integer independently selected from 1 to 5.

Still more preferably, in a compound of formula (II), (IIa), (III) or(IIIa), L₁ is a group of formula X₄—X₅, wherein X₄ is linked to P and X₅is linked to X₁, wherein:

X₄ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹— or —NR¹—SO₂—; and

X₅ is a group of formula —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is aninteger from 6 to 20, preferably from 8 to 18, and p is an integer from1 to 5.

A particularly suitable class of compounds for use as chemoprobes in themethods of the invention is a compound of formula (II), preferably offormula (IIa), or a salt thereof,

wherein:

P is a tag or label;

D is cyclyl or —(C₁₋₄ alkylene)-cyclyl, wherein cyclyl and the cyclylmoiety in the —(C₁₋₄ alkylene)-cyclyl are each optionally substituted,preferably D is cycloalkyl, benzocycloalkyl, heterocycloalkyl or —(C₁₋₄alkylene)-cyclyl, wherein the cycloalkyl, the benzocycloalkyl, theheterocycloalkyl and the cyclyl moiety in the —(C₁₋₄ alkylene)-cyclylare each optionally substituted, and more preferably D is optionallysubstituted cycloalkyl, optionally substituted heterocycloalkyl,—CH₂-heterocycloalkyl wherein the heterocycloalkyl in the—CH₂-heterocycloalkyl is optionally substituted, or —CH₂-heteroarylwherein the heteroaryl in the —CH₂-heteroaryl is optionally substituted;

L₁ is a group of formula X₄—X₅, wherein X₄ is linked to P and X₅ islinked to X₁, wherein:

X₄ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹— or —NR¹—SO₂—;

X₅ is a group of formula —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is aninteger from 6 to 20, preferably from 8 to 18, and p is an integer from1 to 5;

X₁ is —NR¹—C(═O)—, —C(═O)—NR¹—, —SO₂—NR¹— or —NR¹—SO₂—, preferably X₁ is—NHC(═O)—, —C(═O)NH—, —SO₂—NH— or —NH—SO₂—, more preferably X₁ is—NHC(═O)— or —C(═O)NH—, and still more preferably X₁ is —NHC(═O)—; andX₂ is C₁₋₅ alkylene, preferably —(CH₂)₁₋₅—; and more preferably X₁—X₂ isa group of formula

wherein the group is linked to the ring through the carbon atom and toL₁ through the N atom;

each of Z₁ and Z₂ is CH; and

each of s and t is 0.

Still more preferred compounds for use as chemoprobes in the methods ofthe invention is a compound of formula (III), preferably of formula(IIIa), or a salt thereof

wherein:

P is a tag or label;

L₁ is a group of formula X₄—X₅, wherein X₄ is linked to P and X₅ islinked to X₁, wherein:

X₄ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹— or —NR¹—SO₂—;

X₅ is a group of formula —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is aninteger from 6 to 20, preferably from 8 to 18, and p is an integer from1 to 5;

X₁ is —NR¹—C(═O)—, —C(═O)—NR¹—, —SO₂—NR¹— or —NR¹—SO₂—, preferably X₁ is—NHC(═O)—, —C(═O)NH—, —SO₂—NH— or —NH—SO₂—, more preferably X₁ is—NHC(═O)— or —C(═O)NH—, and still more preferably X₁ is —NHC(═O)—; andX₂ is C₁₋₅ alkylene, preferably —(CH₂)₁₋₅—; and more preferably X₁—X₂ isa group of formula

wherein the group is linked to the ring through the carbon atom and toL₁ through the N atom;

each of Z₁ and Z₂ is CH; and

each of s and t is 0.

Throughout this document, divalent chemical groups depicted usingspecific chemical formulae, such as X₁, X₂, X₃ etc are preferablyoriented in the direction in which they are written. As an example, ifin a compound of formula (IIIa) X₁ is —NHC(═O)—, this group ispreferably oriented so that the N atom is bound to L₁ and the C atom isbound to the X₂ group.

In some of the embodiments described above, certain cyclic groups aredefined to be optionally substituted. This means that the group can beunsubstituted or can have one or more substituents. Examples of optionalsubstituents for cyclic groups include, without limitation, thefollowing groups:

C₁₋₄ alkyl, cyclyl, cyclylC₁₋₄ alkyl-, —NH₂, —NR^(a)R^(c), —NO₂, halo,haloC₁₋₄ alkyl, haloC₁₋₄ alkoxy, C₁₋₄ alkyl-O—C₁₋₄ alkyl-, —CN,—C(═O)—NH₂, —C(═O)—NR^(a)R^(c), —NR^(a)—C(═O)—R^(c), —S(═O)—R^(c),—S(═O)₂—R^(c), —NR^(a)—S(═O)₂—R^(c), —S(═O)₂—NH₂, —S(═O)₂—NR^(a)R^(c),—OH, —OR^(c), —C(═O)—R^(c), —C(═O)—OH, —C(═O)—OR^(c), —O—C(═O)—R^(c),—NR^(a)—C(═O)—OR^(c), —O—C(═O)—NR^(a)R^(c), wherein:

each R^(a) and R^(b) are independently selected from hydrogen, C₁₋₄alkyl and haloC₁₋₄ alkyl; and

each R^(c) is independently selected from C₁₋₄ alkyl, haloC₁₋₄ alkyl,cyclyl, cyclylC₁₋₄ alkyl-, and C₁₋₄ alkyl-O—C₁₋₄ alkyl-.

In a compound of formula (I), (II), (IIa), (III) and (IIIa), P is a tagor label. Examples of tags or labels that can be used in said compoundsinclude the tags and labels described in more detail in thecorresponding section below.

In some embodiments, P is a tag. In some embodiments, P is biotin or abiotin derivative.

In some embodiments, P is a label. In some embodiments, P is fluorescentlabel. In some embodiments, P is fluorescein or a fluoresceinderivative.

Preferably, P is biotin or a biotin derivative.

Thus, in a compound of formula (II), (IIa), (III) or (IIIa), it ispreferred that X₄ is —C(═O)—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹— or —NR¹—SO₂—; and that P is biotin or a biotin derivative.

Accordingly, it is particularly preferred that P—X₄— is selected fromthe groups listed below:

wherein R¹ is hydrogen or C₁₋₄ alkyl, and preferably R¹ is H.

It is even more preferred that in a compound of formula (II), (IIa),(III) or (IIIa) P—X₄— is selected from the groups listed below:

wherein R¹ is hydrogen or C₁₋₄ alkyl, and preferably R¹ is H.

It is still even more preferred that in a compound of formula (II),(IIa), (III) or (IIIa) P—X₄— is

wherein R¹ is hydrogen or C₁₋₄ alkyl, and preferably R¹ is H (as, e.g.,in the Examples 1.1 to 1.6).

It is to be understood that the present invention specifically relatesto each and every combination of features or embodiments describedabove, including any combination of general and/or preferredfeatures/embodiments.

In particular, the invention specifically relates to all combinations ofpreferred features/embodiments (including all degrees of preference) ofthe compounds disclosed herein.

Particularly preferred compounds of the invention for use as chemoprobesare the compounds listed below, and salts thereof

Preferably, the compounds of formula (I) (where Z is Z1, Z2, Z3a, Z3b),(II) and (III), including the compounds listed above, have the (S)configuration on the C atom in the cyclopropyl to which the phenyl ringis linked and the (R) configuration in the C atom in the cyclopropyl towhich the N atom is linked.

Most preferred compounds for use as chemoprobes in the methods of theinvention are the compounds listed below, and salts thereof:

A particularly preferred compound is the compound of formula

and salts thereof. This compound is named asN-(39-(4′-((1S,2R)-2-(((trans)-4-aminocyclohexyl)amino)cyclopropyl)-[1,1-biphenyl]-3-yl)-37-oxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azanonatriacontyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide,and is described in example 1.6.

Unless defined for a specific term otherwise, in the definitions of thecompounds of the invention of formula (I), (II), (IIa), (III) and(IIIa), the following definitions apply, when applicable:

As used herein, the term “cyclyl” refers any kind of ring regardlessfuseness, aromaticity and hetero-carbo nature. Examples of cyclylinclude aryl, heterocyclyl (including heteroaryl and heterocycloalkyl),benzocycloalkyl and cycloalkyl group as defined herein.

As used herein, the term “aryl” refers to a carbocyclic aromatic systemcontaining one ring, or two or three rings fused together wherein thering atoms are all carbon. The term “aryl” includes, but is not limitedto groups such as phenyl, naphthyl, or anthracenyl. The term “monocyclicaryl” refers to phenyl. An aryl group is preferably phenyl.

As used herein, the term “cycloalkyl”, unless otherwise specified,refers to a saturated monocyclic, bicyclic or tricyclic group whereinthe ring atoms of the cyclic system are all carbon and wherein eachcyclic moiety contains from 3 to 12 carbon atom ring members. Examplesof cycloalkyl groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or adamantyl. Apreferred group of cycloalkyls is monocyclic C3-7 cycloalkyl.

As used herein, the term “heteroaryl” refers to a 5- to 6-memberedunsaturated monocyclic ring, or a fused bicyclic or tricyclic ringsystem in which the rings are aromatic and in which at least one ringcontains at least one heteroatom selected from the group consisting ofO, S, and N. Preferred heteroaryl groups are 5- to 6-membered monocyclicor 9- to 10-membered bicyclic heteroaryl groups. Examples of heteroarylgroups include, but are not limited to, pyridinyl, imidazolyl,imidazopyridinyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl,tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl,oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl,benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,quinazolinyl, quinoxalinyl, naphthyridinyl, or furopyridinyl.

As used herein, the term “heterocyclyl” or “heterocycle” each refer to asaturated, partially unsaturated, or fully unsaturated monocyclic,bicyclic, or tricyclic heterocyclic group containing at least oneheteroatom as a ring member, wherein each said heteroatom may beindependently selected from the group consisting of nitrogen, oxygen,and sulfur wherein the nitrogen or sulfur atoms may be oxidized (e.g.,—N═O, —S(═O)—, or —S(═O)₂—). Additionally, 1, 2, or 3 of the carbonatoms of the heterocyclyl may be optionally oxidized (e.g., to give anoxo group or ═O). One group of heterocyclyls has from 1 to 4 heteroatomsas ring members. Another group of heterocyclyls has from 1 to 2heteroatoms as ring members. One group of heterocyclyls has from 3 to 8ring members in each ring. Yet another group of heterocyclyls has from 3to 7 ring members in each ring. Again another group of heterocyclyls hasfrom 5 to 6 ring members in each ring. “Heterocyclyl” is intended toencompass a heterocyclyl group fused to a carbocyclyl or benzo ringsystems. Examples of heterocyclyl groups include, but are not limitedto, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino,morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl,azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl,oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinylimidazolinyl, or imidazolidinyl. Examplesof heteroaryls that are heterocyclyls include, but are not limited to,pyridinyl, imidazolyl, imidazopyridinyl, pyrimidinyl, pyrazolyl,triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl,indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl,isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl,thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,naphthyridinyl, or furopyridinyl.

As used herein, the term “heterocycloalkyl” refers to a heterocyclylgroup that is not fully unsaturated e.g., one or more of the ringssystems of a heterocycloalkyl is not aromatic. Preferably, theheterocyclyl group is fully saturated and contains from 1 to 4heteroatoms selected from N, O and S, more preferably contains at least1 N atom. In some embodiments, the heterocycloalkyl is a fully saturated3- to 7-membered monocyclic or 7- to 15-membered polycyclic ring (whichcontains preferably two or three rings in the ring system, which can befused, bridged and/or spiro rings), and wherein the heterocycloalkylcontains from 1 to 4 heteroatoms selected from N, O and S. Examples ofheterocycloalkyls include azetidinyl, piperazinyl, morpholinyl,piperidinyl, or pyrrolidinyl.

As used herein, the term “benzocycloalkyl” refers to a cycloalkyl asdefined herein above, which cycloalkyl is fused to a phenyl ring (i.e.,shares two adjacent ring carbon atoms with a phenyl ring). Non-limitingexamples of benzocycloalkyls are indyl (i.e., 2,3-dihydro-1H-indenyl orbenzocyclopentyl), 1,2,3,4-tetrahydronaphtyl (i.e., tetralinyl orbenzocyclohexyl), or benzocycloheptyl. It is preferred that thebenzocycloalkyl is attached to the remainder of the molecule via itscycloalkyl moiety (and not via its phenyl ring moiety).

The compounds of the present invention contain one or more basicnitrogens and may, therefore, form salts with organic or inorganicacids. Examples of these salts include: salts with inorganic acids suchas hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid,perchloric acid, sulfuric acid or phosphoric acid; and salts withorganic acids such as methanesulfonic acid, thfluoromethanesulfonicacid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,fumaric acid, oxalic acid, acetic acid, maleic acid, ascorbic acid,citric acid, lactic acid, tartaric acid, malonic acid, glycolic acid,succinic acid and propionic acid, among others. The salts of a compoundof the invention, for example a compound of formula I, II, IIa, III orIIIa, can be obtained during the final isolation and purification of thecompounds of the invention or can be prepared by treating the compoundwith a sufficient amount of the desired acid to give the salt in aconventional manner. The salts of the compounds of the invention can beconverted into other salts by ion exchange using ionic exchange resins.

The compounds of formula I, II, IIa, III or IIIa and their salts maydiffer in some physical properties but they are equivalent for thepurposes of the present invention. All salts of the compounds of formulaI, II, IIa, III or IIIa are included within the scope of the invention.The salts are preferably pharmaceutically acceptable salts. As usedherein, a “pharmaceutically acceptable salt” is intended to mean a saltthat retains the biological effectiveness and/or properties of theparent compound (i.e. the free acid or free base, as applicable) andthat is not biologically or otherwise undesirable. Pharmaceuticallyacceptable salts include salts formed with inorganic or organic bases,and salts formed with inorganic and organic acids. Pharmaceuticallyacceptable salts are well known in the art. Exemplary pharmaceuticallyacceptable salts include those salts prepared by reaction of thecompounds of the present invention with a mineral or organic acid, suchas hydrochlorides, hydrobromides, sulfates, pyrosulfates, bisulfates,sulfites, bisulfites, phosphates, monohydrophosphates,dihydrophosphates, metaphosphates, pyrophosphates, chlorides, bromides,iodides, nitrates, acetates, propionates, decanoates, caprylates,acrylates, formates, isobutyrates, caproates, heptanoates, propiolates,oxalates, malonates, succinates, suberates, sebacates, fumarates,maleates, butyne-1,4 dioates, hexyne-1,6-dioates, benzoates,chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates,methoxybenzoates, phthalates, sulfonates, xylenesulfonates,phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates,gamma-hydroxybutyrates, glycollates, tartrates, methane-sulfonates,ethane-sulfonates, propanesulfonates, benzenesulfonates,toluenesulfonates, trifluoromethansulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, mandelates, pyruvates, stearates, ascorbates,or salicylates. When the compounds of the invention carry an acidicmoiety, suitable pharmaceutically acceptable salts thereof may includealkali metal salts, e.g. sodium or potassium salts; alkaline earth metalsalts, e.g. calcium or magnesium salts; and salts formed with suitableorganic ligands such as ammonia, alkylamines, hydroxyalkylamines,lysine, arginine, N-methylglucamine, procaine and the like. Thepharmaceutically acceptable salts of the present invention can beprepared from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. For example, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in a suitablesolvent.

The compounds of the present invention may form complexes with solventsin which they are reacted or from which they are precipitated orcrystallized. These complexes are known as solvates. As used herein, theterm solvate refers to a complex of variable stoichiometry formed by asolute (a compound of formula I, II, IIa, III or IIIa, or a saltthereof) and a solvent. Examples of solvents include pharmaceuticallyacceptable solvents such as water, ethanol and the like. A complex withwater is known as a hydrate. Solvates of compounds of the invention (orsalts thereof), including hydrates, are included within the scope of theinvention

The compounds of formula I, II, IIa, III or IIIa may exist in differentphysical forms, i.e. amorphous and crystalline forms. Moreover, thecompounds of the invention may have the ability to crystallize in morethan one form, a characteristic which is known as polymorphism.Polymorphs can be distinguished by various physical properties wellknown in the art such as X-ray diffraction pattern, melting point orsolubility. All physical forms of the compounds of formula I, II, IIa,III or IIIa, including all polymorphic forms (“polymorphs”) thereof, areincluded within the scope of the invention.

Some of the compounds of the present invention may exist as severaloptical isomers. Optical isomers can be resolved by conventionaltechniques of optical resolution to give optically pure isomers. Thisresolution can be carried out on any chiral synthetic intermediate or onproducts of formula I, II, IIa, III or IIIa. Optically pure isomers canalso be individually obtained using enantiospecific synthesis. Thepresent invention covers all individual isomers as well as mixturesthereof (for example racemic mixtures), whether obtained by synthesis orby physically mixing them.

The KDM1A chemoprobes for use in the methods of the invention describedbelow must exhibit high affinity to KDM1A and selectivity versus otherFAD-dependent monoamine oxidases, such as MAO-A, MAO-B, SMOX, or IL4I1.Importantly, the KDM1A chemoprobes should exhibit high selectivity vsMAO-A and MAO-B, a likely off-target for cyclopropylamine-containingKDM1A inhibitors.

Preferably, the KDM1A chemoprobe has an IC50 against KDM1A below 1 mcM,more preferably below 500 nM.

Preferably, KDM1A chemoprobes should exhibit a selectivity of at least10-fold for KDM1A over other FAD-dependent monoamine oxidases, morepreferably of at least 30-fold for KDM1A over other FAD-dependentmonoamine oxidases, still more preferably of at least 50-fold for KDM1Aover other FAD-dependent monoamine oxidases. Selectivity of at leastX-fold means for example that the IC50 value for KDM1A is at leastX-fold lower than the IC50 value for MAO-A, MAO-B and otherFAD-dependent monoamine oxidases as determined for example usingbiochemical assays.

Different methods are described in the literature that permit to assessKDM1A, MAO-A, MAO-B, SMOX and IL4I1 inhibition. These methods includeassessment of binding to the FAD cofactor by UVNIS spectrometry, massspectrometry, enzymatic assays to detect H₂O₂ or formaldehyde,byproducts of substrate demethylation, alphaLlSA assays to measureH3K4me2/1 demethylation. For example, KDM1A inhibitory activity of thechemoprobes can be determined using the method disclosed in Example 2.The specificity of the chemoprobes for KDM1A over other FAD-dependentenzymes can be tested for example using the methods provided in Example4. The data provided in Example 2 show that the chemoprobes of Examples1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 exhibit potent KDM1A-inhibitoryactivity, with IC50 values around or below 200 nM in all instances.Chemoprobe of example 1.6 (i.e.N-(39-(4′-((1S,2R)-2-(((trans)-4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)-37-oxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azanonatriacontyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide,and salts thereof) was found to be very selective for KDM1A over otherFAD-dependent enzymes as shown by the data provided in Example 4.

The inventors have identified chemoprobes as disclosed herein and in theappended examples, which exhibit potent and selective KDM1A inhibitoryactivity, as shown by the results in the appended Examples. This makesthem particularly suitable for use as chemoprobes in the methodsaccording to the invention, particularly in methods to determine targetengagement of very potent KDM1A inhibitors like ORY-1001. Chemoprobes ofExample 1.1 (i.e.N-(39-(4′-((trans)-2-(((trans)-4-aminocyclohexyl)amino)cyclopropyl-[1,1′-biphenyl]-3-yl)-37-oxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azanonatriacontyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide,and salts thereof) and specially Example 1.6 (i.e.N-(39-(4′-((1S,2R)-2-(((trans)-4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)-37-oxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azanonatriacontyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide,and salts thereof) are particularly preferred.

Tags and Labels

Tags: as defined above, herein tag will be used to describe a moietythat can be incorporated into a molecule, for example into a KDM1Achemoprobe, that can be recognized and used in capture, recovery,isolation, purification or detection procedure of that molecule or ofcomplexes involving that molecule.

Preferentially, tags are small compounds that minimally disturb thefunctionalities, including the binding affinity and selectivity, of themolecule they are incorporated in.

Examples of Suitable Tags are

-   -   Biotin derivatives, which may be recognized by avidin,        streptavidin or streptacin; and    -   Digoxigenin derivatives, which may be recognized by an        anti-digoxigenin antibody.

Examples include:

-   -   Haloalkanes tags, that can be recognized by Halo Tag®        technology, are also suitable tags.

Tags may also be peptidic in nature, examples of peptide tags that canbe incorporated into the chemoprobe/corresponding capture agentsinclude, but are not limited to

-   -   AviTag, SBP, Strep-tag/avidin, streptavidin, streptacin    -   Calmodulin-tag/calmodulin    -   polyglutamate tag/anion-exchange resin such as Mono-Q (EEEEEE)    -   E-tag/anti E-tag antibody    -   FLAG-tag/anti FLAG antibody    -   HA-tag/anti HA antibody    -   polyhistidine tag e.g. 5-10 histidines such as His-tag (HHHHHH)        bound by a nickel or cobalt chelate    -   Myc-tag/anti-Myc antibody    -   TC tag/FlAsH and ReAsH biarsenical compounds (CCPGCC)    -   V5 tag/V5 tag antibody    -   VSV-tag NSV-tag antibody    -   Myc tags/antiMyc tag antibody    -   (poly) histidine tags/nickel and cobalt sepharose or agarose        functionalised with a chelator, such as iminodiacetic acid        (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and        carboxylmethylaspartate (Co-CMA)

Tags may also be

-   -   synthetic oligonucleotides including PNA (protein nucleic acid),        (modified) DNA, (modified) RNA tags/complementary natural or        synthetic PNA, (modified) DNA, (modified) RNA oligonucleotides.        Oligonucleotide tags may be recognized by other oligonucleotides        with complementary (base paring) sequence.

Label: as defined above, herein label will be used to describe a moietythat can be incorporated into a molecule, for example into a KDM1Achemoprobe or detection agent, and that directly gives rise to a signal,for example a fluorescent, bioluminescent, isotope, mass spectrometrylabel. Preferentially, labels will be small (MW<1000), and shouldminimally disturb the functionalities, including the binding affinityand selectivity of the molecule they are incorporated in.

Examples of Fluorescent labels include Alexa Fluor, BODIPY®,Fluorescein, Oregon Green®, Rhodamine Green™, Long wavelengthRhodamines, Texas Red®, Coumarins, Pyrenes, Pyridioxazoles, naphtalenes,Dapoxyl® or Bimane, such as the groups listed below:

A label can also be used as a tag if a suitable capture or detectionagent is available, for example a fluorescein moiety can be recognizedand captured by an anti-fluorescein antibody.

Capture agent: as defined above, this term is used to refer to an agentthat recognizes and binds the tag and that can be used to capture thechemoprobe or chemoprobe complexes from their crude biological source.In this invention, the capture agent can also capture KDM1A or KDM1Acontaining complexes from their crude biological source. The capturereagent can be coupled to a surface. The capturing process can be director indirect, i.e. the recognition p.e. tag included in the chemoprobecan be captured by a primary capture agent coupled to a surface,recognized by a secondary capture agent that recognizes the firstcapture agent and that is bound to a surface. Suitable capture agentsinclude antibodies or proteins with a high selectivity and affinity forthe tag in KDM1A chemoprobe and for the KDM1A chemoprobe boundcomplexes, or with a high selectivity and affinity for KDM1A or KDM1Acontaining complexes.

Examples of suitable capture agents for a specific tag incorporated intothe KDM1A chemoprobe are listed above.

Suitable capture agents to capture KDM1A or KDM1A containing complexespreferentially include high specificity/high affinity antibodies orantibody fragments to KDM1A or to KDM1A containing complexes.

Antibodies can be commercially available or can be produced orsynthesized by any known method.

A specially suitable tag for the KDM1A chemoprobe is biotin and thecorresponding capture agent is streptavidin.

Surface: as mentioned above, the capture agent can be coupled to asurface. This can be any suitable surface or solid support includingmicro or nanobeads, microtiter plates, nanostnngs, nanowires, biofilms,polymers or any other suitable matrix.

Detection agent: as defined above, herein a detection agent is an agentthat recognizes and binds a tag and can be used to detect a tagincorporated in the KDM1A chemoprobe and can be used to detect KDM1Achemoprobe bound complexes. Alternatively, it can be an agent that canbe used to detect KDM1A or KDM1A containing complexes in a sample.

Detection can be direct or indirect, amplified or non-amplified,mediated by a single molecule or a mix of molecules, but invariablyincludes a moiety that generates a signal that can be visualized ormeasured.

The detection agent may be used for direct detection of the KDM1Achemoprobe and chemoprobe bound complexes; or KDM1A or KDM1A containingcomplexes, and directly include the signal generating moiety.Alternatively a primary detection agent may bind the KDM1A chemoprobeand chemoprobe bound complexes; or KDM1A or KDM1A containing complexes,the primary detection agent may be detected by a secondary detectionagent which includes the signal generating moiety.

Said detection agent may include a fluorescent, luminescent,colorimetric, isotope or mass spectrometry label. Alternatively, saiddetection agent may include a synthetic oligo nucleotide, that can beused to generate a signal using proximity ligation assay technology.

Alternatively, said detection agent may include an enzyme, for examplehorse radish peroxidase or alkaline phosphatase, that transforms asubstrate in a colored, fluorescent or luminescent compound. Examples ofsuch enzymes include Horseradish Peroxydase (HRP), Alkaline phosphatase(AP), Glucose oxidase and β-galactosidase enzyme. Colorimetric detectionis based on conjugation with the Horseradish Peroxydase (HRP), Alkalinephosphatase (AP), Glucose oxidase or β-galactosidase enzyme, followed byincubation with the appropriate chromogenic reporter (respectively,3,3′-diaminobenzidine, combination of nitro blue tetrazolium chlorideand 5-bromo-4-chloro-3-indolyl phosphate, Nitro blue tetrazoliumchloride or 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside).Chemiluminescent detection systems are based on the conversion,catalyzed by the HRP enzyme, of luminol to 3-aminophthalate, aluminescent moiety. Examples of substrates for Horseradish Peroxydase(HRP) include:

Said enzyme may be coupled directly to the agent that recognizes a tagincorporated in a KDM1A chemoprobe or KDM1A chemoprobe bound complexes,or it may be coupled to a secondary agent that recognizes said primaryagent. Likewise, said enzyme can be coupled directly to an agent thatrecognizes KDM1A or KDM1A containing complexes, or it may be coupled toa secondary agent that recognizes said primary agent.

Alternatively, said primary detection agent can also incorporate a tag,detected by a secondary detection agent that incorporates a label or amoiety that generates a signal that can be visualized or measured.

Said (primary) detection agent has a high selectivity and affinity forthe tag in KDM1A chemoprobe and for the KDM1A chemoprobe boundcomplexes, or for KDM1A or KDM1A containing complexes. Preferably, said(primary) detection agent comprises a binding molecule like an antibodyor binding protein or other macro(molecule) (e.g. aptamer, affimer, etc)that recognizes the tag incorporated into the KDM1A chemoprobe, or KDM1Aor KDM1A containing complex; with high specificity and high affinity.Herein, an antibody is preferentially a monoclonal antibody, or a singlechain construct like a recombinant ScFv, Fabs or fusions proteinsthereof. The antibody or protein antibody used to detect KDM1A or theKDM1A containing complex should be directed against an epitope which isreadily accessible. Accessibility of the epitope can be modeled insilico, and tested in vitro using immunoprecipitation (IP) as describedin the examples. Suitable KDM1A antibodies are disclosed in theexamples; alternative antibodies can also be identified, following thestrategy disclosed in the examples to identify said suitable antibodies.Alternative antibodies can also be raised against accessible epitopesfrom KDM1A-interacting factors that are part of the KDM1A complex.RCOR1, HDAC1 and HDAC2 are examples of such factors closely interactingwith KDM1A.

Preferably, the detection or capture of the KDM1A bound chemoprobe orKDM1A bound chemoprobe containing complexes by a first detection orcapture agent does not interfere with the detection of KDM1A or KDM1Acontaining complexes by a second detection agent, nor does the detectionor capture of KDM1A or KDM1A containing complexes by a first detectionor capture agent interfere with the detection of KDM1A or KDM1Acontaining complexes by a second detection agent.

The detection system: herein we will refer to a detection system whenmultiple detection agents are required to act in trans and in closeproximity to generate a signal; p.e. the Förster/Resonance EnergyTransfer (FRET), AlphaLISA, DELFIA, and proximity ligation assaytechnologies (protein PCR). In FRET, AlphaLISA and DELFIA a firstdetection agent will function as a donor that absorbs energy andgenerates an energy transfer (FRET) or oxygen singlet transfer(AlphaLISA, DELFIA) to an acceptor and provoke it to emit a signal at aspecific, lower wavelength. In one embodiment of the invention describedherein, a first detection agent is designed to recognize and bind to theKDM1A chemoprobe containing complexes, and a second detection agent tobind the KDM1A enzyme or to the KDM1A containing complex, to detect freeKDM1A. In another embodiment, in addition to detect free KDM1A, twodifferent detection agents that bind to different sites on KDM1A or theKDM1A containing complex are used to detect total KDM1A. Stillalternatively, two KDM1A detection agents that bind to KDM1A or theKDM1A containing complexes and a third detection agent that binds to theKDM1A chemoprobe can be used. One KDM1A detection agent functions as adonor, and the other KDM1A detection agents and the KDM1A chemoprobedetection agent function as acceptors emitting at different wavelengths,allowing for simultaneous detection of free and total KDM1A. In theproximity ligation assay technology, the different detection agentscarry nucleic acids that will anneal with a connector nucleic acid, beligated and form an amplifyable fragment that can be detected using realtime quantitative PCR. Again, the detection agents can detect the KDM1Achemoprobe containing complexes and the KDM1A protein, and can be usedto detect free and total KDM1A independently or in a multiplex reactionin homogeneous or heterogeneous assays.

The tags, labels, capture and detection agents, enzymes, substrates andother tools and techniques described above are just to illustrate waysto carry out the invention and are not intended to be limiting themethods of the invention in any way.

Target Engagement Assays

The chemoprobes of the invention can be used in methods to determinetarget engagement of a KDM1A inhibitor, as described in more detailherein. The methods according to the invention allow for a directassessment of target occupation by KDM1A inhibitors, as opposed tomethods generally used so far, which are based on indirectdeterminations of KDM1A engagement, typically through the analysis ofchanges caused by said KDM1A inhibitors in the level of histone markstargeted by KDM1A or in gene expression.

The methods of the invention are, inter alia, based on the directquantification of target engagement via measuring free KDM1A levels bymeans of chemoprobe “tagging” of the free KDM1A enzyme. As previouslydefined herein, free KDM1A means enzymatically active KDM1A present in asample not bound by a KDM1A inhibitor. “tagging” is used herein in abroad sense, covering the use of both tags and labels as definedpreviously.

In cells, tissues, organs or subjects treated with a KDM1A inhibitor, aportion of the enzymatically active KDM1A enzyme pool will be bound(occupied) by the KDM1A inhibitor, which portion may change according tothe (KDM1A inhibitory) potency of the compound and concentration/doseused. Free KDM1A levels can thus be used to determine how much KDM1Aenzyme is bound by the KDM1A inhibitor, i.e. the target engagement bysuch inhibitor can be determined by the methods described herein. Thechemoprobe-based methods for determining free KDM1A levels according tothe invention are thus useful to assess target engagement and to assessthe pharmacodynamics of KDM1A inhibitors, for example in clinical trialsor in clinical practice.

Accordingly, in one aspect, the invention provides a method fordetermining a level of free KDM1A in a sample or subject, wherein saidmethod comprises

-   -   (i) contacting or exposing KDM1A to a chemoprobe, wherein said        chemoprobe is a compound as defined above and in the appended        claims; and    -   (ii) determining said level of free KDM1A employing said        chemoprobe in said sample or subject.

Methods for determining free KDM1A levels are provided below and in theappended examples.

In another aspect, the invention provides a method for in vitrodetermining a level of free KDM1A in a sample,

-   -   wherein said method comprises    -   (i) contacting or exposing KDM1A to a chemoprobe, wherein said        chemoprobe is a compound as defined above or in the appended        claims; and    -   (ii) determining said level of free KDM1A employing said        chemoprobe in said sample.

In vitro, when used in relation to the methods of the invention todetermine free KDM1A level and to determine target engagement, means, asdefined previously, outside a living organism (which can be for examplea human), i.e. the method is not directly performed on said livingorganism. It thus includes for example methods implemented using samplestaken from a living organism (for example a human) that has received invivo treatment with a KDM1A inhibitor, which samples are referred to as“ex vivo” samples in the examples section. As illustrated in theExamples, methods to determine free KDM1A levels using chemoprobes ofthe invention may be performed in a variety of settings and sampletypes, including human samples.

As illustrated in the appended examples, free KDM1A levels may bedetermined as shown in Example 5 using a KDM1A chemoprobe-based ELISAassay employing a chemoprobe of formula (I) which contains a biotin tag(as P group) to capture free KDM1A, and using different detectiontechniques, like luminescence or colorimetric (see Example 5.2 and 5.3).This method to determine free KDM1A may also be successfully implementedto determine free KDM1A levels in an homogeneous assay format usingAlphaLISA technology, as shown for instance in Example 6 and FIG. 6A.

The chemoprobe-based method described in Example 5.2 was further used todetermine free KDM1A levels in PBMCs isolated from human blood fromhealthy volunteers, as described in Example 5.2 and FIG. 5B. This showsthat the chemoprobes and chemoprobe-based methods of the invention cansuccessfully be used in human samples to determine free KDM1A levels,and accordingly to determine target engagement of KDM1A inhibitors inhumans, as also described in more detail below.

As illustrated in Example 7, free KDM1A levels may be determined usingthe chemoprobe-based methods of the invention in cells treated withKDM1A inhibitors. In particular, in example 7, free KDM1A levels weredetermined in MV(4;11) human leukemia cells treated with several KDM1Ainhibitors (or vehicle), as described in more detail in Example 7.3 andFIGS. 7 and 8A, as well as in other cell lines, including THP-1 humanleukemia cells (see Example 7.3 and FIG. 8B), as well as in solid tumorcell lines like LNCap human prostate cancer cells (see Example 7.3 andFIG. 8C). The free KDM1A levels as determined therein may then be usedto determine target engagement, following the methods described in moredetail below. The results obtained in Example 7 showed a dose-responsecellular target engagement in accordance with the in vitro KDM1Ainhibitory activity of the tested KDM1A inhibitors, further showing thatthe chemoprobes and chemoprobe-based methods according to the inventioncan be used to provide reliable determinations of free KDM1A levels andof KDM1A target engagement by KDM1A inhibitors.

In particular, Example 8 and 9 further illustrate that free KDM1A levelsmay be determined using the chemoprobe-based methods of the invention insamples taken from animals that had been administered in vivo withsingle or repeated doses of a KDM1A inhibitor. For example, as describedin Example 8, free KDM1A levels were determined in PBMCs and lungsamples from rats treated with increasing doses of ORY-1001 or vehicle,and these levels were used to determine target engagement (as describedbelow); the corresponding results are shown in FIGS. 9A and 9B.Additional examples showing the measurement of free KDM1A levels inanimals treated with single or multiple doses of ORY-1001 are disclosedin Example 9. Free KDM1A levels were further determined in brain samplesfrom mice from an experimental Alzheimer's disease model treated withanother KDM1A inhibitor, Compound C (as further described in Example 8),or vehicle, which were used to determine target engagement of Compound Cin the brain (see results in FIG. 9C); the free KDM1A leveldeterminations and target engagement measurements using the methods ofthe invention confirmed that this compound enters the brain and inhibitsKDM1A in the brain.

As illustrated in Example 9, the chemoprobes and methods of theinvention may be used to determine free KDM1A levels in PBMC samplesfrom human subjects which have been administered a KDM1A inhibitor inthe context of a clinical trial. In particular, as described in Example9, in a Phase I clinical trial to determine the safety, tolerability andpharmacokinetics of the KDM1A inhibitor designated as Compound C (asdescribed in the Examples), samples were taken from each volunteerbefore and at different time points after administration of a singledose of the KDM1A inhibitor, and free KDM1A levels were determined insaid samples, and used to determine target engagement, as describedbelow.

The methods to determine free KDM1A levels according to the inventionusing a KDM1A chemoprobe to “tag” free KDM1A are not limited to theparticular embodiments described in the examples. While the methods havebeen implemented using ELISA (or AlphaLISA) platforms, as particularlyELISA is a technology widely implemented in research labs and clinicalanalysis laboratories, the skilled person will readily recognize thatthe chemoprobe-based methods to determine free KDM1A protein levels maybe implemented using other technologies, as outlined for example in thesections Detection agents and Detection systems above.

For example, other differently tagged/labeled KDM1A chemoprobes offormula (I) (or of formula II or III) may be used, where biotin isreplaced with another tag or label P as defined above in relation to acompound of formula (I). In the section entitled Tags and Labels above,other examples of tags and labels that can be used in the chemoprobes ofthe invention instead of biotin are mentioned, as well as correspondingsuitable methods to capture/detect said tags/labels in the chemoprobeand KDM1A-chemoprobe bound complexes, e.g. to determine free KDM1Alevels. Likewise, other chemoprobes containing a different KDM1Ainhibitor radical Z may be used. Chemprobes as described in Examples1.1, 1.2, 1.3 and 1.6, which correspond to a compound of both formula(I), (II) and (III), are based on the KDM1A inhibitor known as ORY-1001,whereas chemoprobes of examples 1.4 and 1.5, which correspond to acompound of both formula (I) and (II), are based on two other KDM1Ainhibitor Z radicals. Alternative suitable chemoprobes may be preparedand used based on other KDM1A inhibitors, i.e. containing other Zradicals, preferably wherein Z is a radical of an irreversible KDM1Ainhibitor. Examples of KDM1A inhibitors that may be used to prepareKDM1A chemoprobes are disclosed in the section KDM1A chemoprobes, above.Additional examples of KDM1A inhibitors are discussed in the sectionKDM1A inhibitors below, and they may also be used to generate KDM1Achemoprobes. The resulting KDM1A chemoprobe should be exhibitingappropriate KDM1A inhibitory potency and selectivity versus othertargets for use in the methods of the invention, as discussed aboveunder the KDM1A chemoprobe section.

In some embodiments, the above methods further comprises to determine alevel of total KDM1A in said sample. For example, total KDM1A levels maybe used for sample normalization, in order to correct for differences inthe amount of KDM1A enzyme present in a sample, when comparing resultsobtained in different samples.

For example, total KDM1A levels may be determined as illustrated in theExamples. For example, as described in Examples 5 and 6, total KDM1Alevels may be determined using a sandwich ELISA (see Example 5 and FIG.3A, showing a schematic representation of the sandwich ELISA) or in acorresponding AlphaLISA format (see Example 6) using two differentantiKDM1A antibodies, which were selected using the criteria describedin more detail in Example 5.1 and in the section Antibodies below. Otheralternative antiKDM1A antibodies may be used, which may be selected asdescribed in said Antibodies section. While an ELISA-based method suchas for example the one described in the Examples is advantageous, asELISA technology platforms are widely implemented in research andclinical analysis labs, alternative methods to determine the totalamount of KDM1A protein present in a sample may be used, for example themethods outlined above under the sections Detection agent and Detectionsystem, as will be apparent to those skilled in the art.

A level can be expressed as needed in different units (p.e. RelativeLuminescence Units or RLU, Absorbance Units or AU, Fluorescence Units orFU, etc) in function of the technology used to obtain a signal, as longas there is a linear relationship between the amount of signal and theamount of free or total KDM1A in a sample (i.e. a dilution curve showsdetection is in the linear range). The level can also be expressed as aconcentration (amount), if a calibration curve was established fromsamples containing known amounts of total or free KDM1A protein and theconcentration (amount) can be determined through interpolation.

The raw signals obtained from the measuring equipment can be subject todata processing. Data processing that is generally applied issubtraction of background signal, outlier elimination using establishedcriteria like the Grubbs' test, Chauvenet's criterion, the MMS-test foroutliers in linear regression, Peirce's criterion and business rules.The mean or median signal can be calculated, as well as the error on themeasurement.

In some embodiments, the above methods further comprise determiningtarget engagement of a KDM1A inhibitor in said sample.

In some embodiments, the determination of said target engagementcomprises calculating the ratio between free KDM1A level in the sampleand the free KDM1A level in a reference sample.

In some embodiments, the determination of said target engagementcomprises calculating the ratio between free KDM1A level and total KDM1Alevel in the sample.

In some embodiments the determination of said target engagementcomprises calculating the ratio A/B, wherein A is the ratio of the freeKDM1A level and the total KDM1A level in the sample and B is the ratioof the free KDM1A level and total KDM1A level in the reference sample.

Target engagement (TE) of a KDM1A inhibitor can be determined in variousways.

In a first method, TE in a sample X (TEx) can be calculated asTE_(X)=1−LEVEL_(Free,X)/LEVEL_(Total, X); wherein LEVEL_(Free,X) is thelevel of free KDM1A in sample X and LEVEL_(Total, X) is the level oftotal KDM1A in sample X. This method of calculation can be used on thecondition that the detection efficiency of free and total KDM1A areequal.

Accordingly, the invention provides a method to determine targetengagement of a KDM1A inhibitor in a sample, wherein said targetengagement is determined as 1 minus the ratio between free KDM1A leveland total KDM1A level in the sample, wherein 1 corresponds to fulltarget engagement and 0 corresponds to absence of target engagement.

In a second method, TE in a sample X (TEx) can also be calculated asTE_(X)=1−LEVEL_(Free,X)/LEVEL_(Free, REF); wherein LEVEL_(Free,X) is thelevel of free KDM1A in sample X and LEVEL_(Free, REF) is the level offree KDM1A in a reference sample. This method can be used on thecondition that the amount of total KDM1A present in sample X and in thereference sample is the same and on the condition that the detectionefficiency of free KDM1A in sample X and in the reference sample is thesame.

Accordingly, the invention provides a method to determine targetengagement of a KDM1A inhibitor in a sample, wherein said targetengagement is determined as 1 minus the ratio between free KDM1A levelin the sample and the free KDM1A level in a reference sample, wherein 1corresponds to full target engagement and 0 corresponds to absence oftarget engagement.

In a third method, TE in sample X can also be calculated asTE_(X)=1−(R_(X)/R_(REF)); whereR _(X)=LEVEL_(Free,X)/LEVEL_(Total,X) and R_(REF)=LEVEL_(Free,REF)/LEVEL_(Total,REF);wherein LEVEL_(Free,X) is the level of free KDM1A in sample X,LEVEL_(Total, X) is the level of total KDM1A in sample X,LEVEL_(Free, REF) is the level of free KDM1A in a reference sample andLEVEL_(Total, REF) is the level of total KDM1A in the reference sample.

This is equivalent to calculating TE usingTE_(X)=1−(R_(Free)/R_(Total)); whereR _(Free)=LEVEL_(Free,X)/LEVEL_(Free,REF and) R_(Total)=LEVEL_(Total,X)/LEVEL_(Total,REF)

The advantage of this calculation method is that it does not require thedetection efficiency of free and total KDM1A to be equal, nor does itrequire that the same amount of total KDM1A is used in every assay,which speeds up the procedure. The formula can be applied when it can beassumed that the detection efficiency of free KDM1A in sample X and thereference sample will be the same, and that the detection efficiency oftotal KDM1A will be the same in sample X and the reference sample. Thisis usually the case when both samples are of the same nature; p.e,extracts of a specific tissue or cell type.

Accordingly, the invention provides a method to determine targetengagement of a KDM1A inhibitor in a sample, wherein said targetengagement is determined as 1 minus the ratio R_(X)/R_(REF), whereinR_(X) is the ratio of the free KDM1A level and the total KDM1A level inthe sample and R_(REF) is the ratio of the free KDM1A level and totalKDM1A level in the reference sample, wherein 1 corresponds to fulltarget engagement and 0 corresponds to absence of target engagement.

In any case, target engagement calculated according to the above methodscan be expressed as a fraction of 1 or in %, for example:TE _(X) (%)=100−((R _(X)(%)/R _(REF)(%))×100); where R_(X)(%)=(LEVEL_(Free,X)/LEVEL_(Total,X))×100 and R _(REF)(%)=(LEVEL_(Free,REF)/LEVEL_(Total,REF)×)100.

Accordingly, the invention provides a method for in vitro determiningtarget engagement of an inhibitor of KDM1A in a sample, wherein saidmethod comprises

-   -   (i) contacting or exposing KDM1A to a chemoprobe of the        invention;    -   (ii) determining a level of free KDM1A employing said chemoprobe        in said sample;    -   (iii) determining a level of free KDM1A in a reference sample;    -   (iv) calculating the ratio between the free KDM1A level in the        sample and the free KDM1A level in the reference sample; and    -   (v) determining target engagement as 1 minus the ratio        calculated in step (iv).

In some embodiments, the method comprises contacting or exposing thesample to said chemoprobe.

In another aspect, the invention provides a method for in vitrodetermining target engagement of an inhibitor of KDM1A in a sample,wherein said method comprises

-   -   (i) contacting or exposing KDM1A to a chemoprobe of the        invention;    -   (ii) determining a level of free KDM1A employing said chemoprobe        in said sample;    -   (iii) determining a level of total KDM1A in said sample;    -   (iv) calculating the ratio between free KDM1A level and total        KDM1A level in the sample; and    -   (v) determining target engagement as 1 minus the ratio        calculated in step (iv).

In some embodiments, the method comprises contacting or exposing thesample to said chemoprobe.

In another aspect, the invention provides a method for in vitrodetermining target engagement of an inhibitor of KDM1A in a sample,wherein said method comprises

-   -   (i) contacting or exposing KDM1A to a chemoprobe of the        invention;    -   (ii) determining a level of free KDM1A employing said chemoprobe        in said sample;    -   (iii) determining a level of total KDM1A in said sample;    -   (iv) determining a level of free KDM1A employing said chemoprobe        in a reference sample;    -   (v) determining a level of total KDM1A in said reference sample;    -   (vi) calculating the ratio A/B, wherein A is the ratio of the        free KDM1A level and the total KDM1A level in the sample and B        is the ratio of the free KDM1A level and the total KDM1A level        in the reference sample; and    -   (vii) determining target engagement as 1 minus the ratio        calculated in step (vi).

In some embodiments, the method comprises contacting or exposing thesample to said chemoprobe.

In some embodiments, the sample has been exposed or contacted to a KDM1Ainhibitor in vitro.

In some embodiments, the sample is obtained from a subject that has beenadministered a KDM1A inhibitor.

In step (i) in the above methods, KDM1A is contacted or exposed to achemoprobe of the invention. Any compound of formula (I), (II), (IIa),(III) or (IIIa) as described herein may be used as chemoprobe in theabove methods. In the appended examples, chemoprobes of formula (I),(II), (IIa), (III) or (IIIa) wherein P is biotin have been used, butother differently tagged/labeled KDM1A chemoprobes of formula (I) (or offormula II, IIa, III or IIIa) may be used, where biotin is replaced withanother tag or label P as defined above in relation to a compound offormula (I). In the section entitled Tags and Labels above, examples oftags and labels that can be used in the chemoprobes of the inventioninstead of biotin are mentioned. Likewise, other chemoprobes containinga different KDM1A inhibitor radical Z other than the ones shown in thechemprobes as described in the Examples may be used. Preferably Z shouldbe a radical of an irreversible KDM1A inhibitor. Examples of KDM1Ainhibitors that may be used to prepare KDM1A chemoprobes are disclosedin the section KDM1A chemoprobes, above. Additional examples of KDM1Ainhibitors are discussed in the section KDM1A inhibitors below, and theymay also be used to generate KDM1A chemoprobes. The resulting KDM1Achemoprobe should exhibit appropriate KDM1A inhibitory potency andselectivity versus other targets for use in the methods of theinvention, as discussed above.

Said contacting or exposing KDM1A to the chemoprobe may be performed bycontacting or exposing the sample (containing KDM1A) in vitro to thechemoprobe, such as by lysing the sample in the presence of thechemoprobe.

Said sample as used in step (i) in the above methods may be a samplethat has been contacted or exposed to a KDM1A inhibitor in vitro, forexample a cell culture like a human cell culture is contacted or exposedto said KDM1A inhibitor in vitro, or a tissue, organ or blood sampleobtained from a subject is contacted or exposed to said KDM1A inhibitorin vitro. The sample may also be a sample that is obtained from asubject (for example a human or animal) that has been administered aKDM1A inhibitor. The sample may be for example a peripheral sample (e.g.PBMCs isolated from blood), biopsy, extracted tissues, extracted tumorand the like.

In step (ii) in the above methods, free KDM1A level in said sample isdetermined using said chemoprobe of the invention. As illustrated in theexamples, free KDM1A level may be determined using a KDM1Achemoprobe-based ELISA assay employing a chemoprobe of formula (I) (orII, IIa, III or IIIa) which contains a biotin tag (as P group) tocapture free KDM1A, and using different detection techniques, likeluminescence or calorimetric. The method used to determine free KDM1Alevels according to the invention using a KDM1A chemoprobe is notlimited however to the particular embodiments described in the examplesand other methods to determine free KDM1A levels may be used, as will beapparent to those skilled in the art. For example, it is possible to useother P tags or labels in the chemoprobe and/or other suitable captureagents (as described for example in the section Capture agents above)and/or other detection methods (as described for example in the sectionsDetection agent and Detection system above).

Some of the above methods comprise a step of determining a level oftotal KDM1A in the sample. As illustrated in the examples, total KDM1Alevels may be determined using a sandwich ELISA (or correspondingAlphaLISA assay) using two different antiKDM1A antibodies. While anELISA-based method such as for example the one described in the Examplesis advantageous, as ELISA technology platforms are widely implemented inresearch and clinical analysis labs, alternative methods to determinethe total amount of KDM1A protein present in a sample may be used, aswill be apparent to those skilled in the art, for example the methodsoutlined above under the sections Detection agent and Detection system.

Some of the above methods comprise a step of determining a level of freeKDM1A or total KDM1A in a reference sample. As defined above, thereference sample may be any tissue, cells, protein extract, recombinantprotein that is chosen to serve as a reference to the sample understudy. A reference sample, or the subject from which the referencesample has been obtained, may have been treated with a vehicle, or mayhave been obtained from the same subject prior to treatment with a KDM1Ainhibitor, or may have been obtained from the same subject when theeffect of the KDM1A inhibitor has ceased completely. Free KDM1A levelsand total KDM1A levels in said reference sample may be determined usingthe same methods to determine free KDM1A levels and total KDM1A levels,respectively, in the sample under study.

The thus determined free and/or total KDM1A levels in a sample and/or ina reference sample may then be used to calculate the ratios as definedabove, which are then used to determine target engagement as describedabove. Target engagement calculated according to the above methods maythen be expressed as a fraction of 1 or in %.

For example, as illustrated in Example 7, target engagement of a KDM1Ainhibitor may be determined as follows: a sample of cells (e.g.MV(4;11)) treated with a KDM1A inhibitor (e.g. DRY-1001) or with vehicle(used as reference sample) is lysed in the presence of a chemoprobe ofthe invention (e.g. example 1.6) and protein extracts are obtained suchas described in Example 7.1, thereby contacting or exposing KDM1A(present in said cell sample) to said chemoprobe, as recited in step (i)above; the level of free KDM1A may be measured in said protein extractcontaining chemoprobe bound to KDM1A using the chemoprobe-based ELISAassay described in Example 5.2, as explained in Example 7.3, thusdeterming a level of free KDM1A employing said chemoprobe in said sampleas recited in step (ii) above; the level of total KDM1A may be measuredin the same protein extract using the sandwich ELISA assay described inExample 5.2, as explained in Example 7.3, thus determing a level oftotal KDM1A in said sample as recited in step (iii) above; sameprocedure may be followed using the sample of cells treated with vehicledescribed above as reference sample to measure free and total KDM1Alevel in said reference sample, thus determining a level of free KDM1Aemploying said chemoprobe in said reference sample as recited in step(iv) above, and determining a level of total KDM1A in said referencesample as recited in step (v); and the obtained values of free and totalKDM1A level in the sample of cells treated with ORY-1001 and in thereference sample (treated with vehicle only) may be then used asdescribed in Example 7.2 and 7.3 to calculate the ratio of freeKDM1A/total KDM1A level in the sample (indicated as sample X in Example7.2), corresponding to A above in step (vi) and the ratio of freeKDM1A/total KDM1A level in the reference sample (indicated as sample REFin Example 7.2), corresponding to B above in step (vi) above and finallyto calculate the ratio NB, as recited in step (vi) above, and thentarget engagement may be calculated as outlined in Example 7.2, wheretarget engagement in the sample (indicated as TEx in Example 7.2)corresponds to 1-NB, thus determining target engagement as 1 minus theratio calculated in step (vi), as recited in step (vii) above. Targetengagement may be expressed as a fraction of 1 or in %, as explained inmore detail above.

As illustrated in the Examples, target engagement of KDM1A inhibitorsmay be determined according to the above methods in a range of sampletypes, including human samples. For example, target engagement for KDM1Ainhibitors, may be determined in cell lines treated with KDM1Ainhibitors, as shown in Example 7, in particular in MV(4;11) leukemiacells (see Example 7.3 and FIGS. 7 and 8A), as well as in other celllines, including THP-1 leukemia cells (see Example 7.3 and FIG. 8B), andLNCap prostate cancer cells (see Example 7.3 and FIG. 8C).

As illustrated in the Examples, the methods of the invention may also beused to determine target engagement in samples taken from animals thathave been administered in vivo with single or repeated doses of a KDM1Ainhibitor. As illustrated in Example 8, target engagement for a KDM1Ainhibitor (for example ORY-1001) may be determined in peripheral samples(for example PBMCs) and tissue (for example lung) samples from subjects(for example rats) treated with increasing doses of the KDM1A inhibitoror vehicle, as described in more detail in Example 8.3 and FIGS. 9A and9B. Example 8 further illustrates that target engagement may bedetermined in further tissue samples, like brain samples; in particular,target engagement was determined in brain samples from SAMP8 mice (anexperimental Alzheimer's disease model) treated with the KDM1A inhibitordesignated as Compound C (a compound that crosses the blood-brainbarrier in the brain, further described in Example 8), as described inExample 8.3 and in FIG. 9C.

As illustrated in the appended Examples, the methods of the inventionmay also be used to determine target engagement for a KDM1A inhibitor inhuman samples. In particular, Example 9 illustrates that targetengagement for a KDM1A inhibitor may be determined in peripheral samples(PBMCs) from humans participating in a clinical trial. As described inExample 9 in more detail, as part of a Phase I clinical trial with theKDM1A inhibitor designated Compound C, whose main objective is todetermine the safety, tolerability and pharmacokinetics of Compound C,cohorts of healthy volunteers have received a single dose of Compound C,or placebo. Samples have been taken from each volunteer at differenttime points and free and total KDM1A levels have been determined in saidsamples and have been used to calculate target engagement for CompoundC, following the method described in Example 7.2.

The hereindescribed KDM1A chemoprobes and methods have been applied toassess target engagement by ORY-1001 and other KDM1A inhibitors, asdescribed in more detail in the Examples, and can be readily used toassess target engagement by other KDM1A inhibitors, such as thecompounds disclosed in the “KDM1A inhibitors” section below. To ensurethat the KDM1A chemoprobe and not residual unbound KDM1A inhibitoroccupies the free KDM1A enzyme, preferentially the IC50 of thechemoprobe should be maximum 10× the IC50 of the KDM1A inhibitor and thechemoprobe should be added in excess.

In some embodiments, the KDM1A inhibitor is an irreversible KDM1Ainhibitor. In some embodiments, the irreversible KDM1A inhibitorinhibits KDM1A through covalent binding to the FAD cofactor. In someembodiments, the KDM1A inhibitor is(trans)-N1-((1R,2S)-2-phenylcyclopropyl)cyclohexane-1,4-diamine, or(−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine.In some other embodiments, the KDM1A inhibitor is4-((4-((((1R,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-1-yl)methyl)benzoicacid, or a salt thereof.

KDM1A target engagement by reversible KDM1A inhibitors that block theenzymatic activity of KDM1A can also be assessed. In this case, a lesspotent KDM1A chemoprobe with appropriate binding kinetics may bepreferred to avoid that the chemoprobe displaces the reversible KDM1Ainhibitor during the analysis procedure.

Alternatively or combined with this approach, the chemoprobe bindingreaction during the analysis procedure may be stopped by addition of anexcess of a potent irreversible KDM1A inhibitor like ORY-1001 atselected timepoints.

In some embodiments, in the above methods the chemoprobe is used tocapture or detect free KDM1A.

In some embodiments, in the chemoprobes of the invention P is a tag andthe chemoprobe is captured or detected by a suitable capture ordetection agent directed against the tag. The nature of the captureand/or detection agent will depend on the tag used. Examples of tags andcorresponding capture or detection agents are explained in more detailunder the section “Tags and labels”.

In some embodiments, the determination of the level of free KDM1Acomprises the use of an antibody specific to an epitope of KDM1A.

In some embodiments, the determination of the level of total KDM1Acomprises the use of a first antibody specific to an epitope of KDM1A tocapture or detect total KDM1A. In some embodiments, the determination ofthe level of total KDM1A comprises the use of a second antibody specificto an epitope of KDM1A to capture or detect total KDM1A, wherein saidepitope of said second antibody is different from said epitope of thefirst antibody.

In some embodiments, the above methods comprise the use of a protein tocapture or detect the tag in the chemoprobe and to determine said levelof free KDM1A. Proteins that can be used are described in detail in the“Tags and labels” section. In one embodiment, such protein isstreptavidin.

When selecting the detection and capture agents for use in the abovemethods, it is desirable that the binding of any of the antibodies tothe target epitopes on KDM1A or the binding of the protein to the tag inthe KDM1A-bound chemoprobe does not interfere with the ability of theremaining antibody(ies) to bind its target epitope on KDM1A or of theprotein to bind the KDM1A-bound chemoprobe.

Antibodies

The criteria for selection of the antibodies to be used in the abovemethods of the invention will depend on the detection technology chosenfor use with the methods of the invention. Factors to be consideredinclude specificity, affinity, proximity and sterical hindrance,including the likeliness of mutual interference of binding of antibodiesor probe, and the probability of the antibody to recognize the nativeenzyme within the protein complexes that contain it.

A suitable pair of antibodies for use in combination with the abovemethods includes an antibody directed against an epitope located at theN-terminal region of KDM1A and an antibody directed against an epitopelocated at the the C-terminal region of KDM1A. In one embodiment, thepair of antibodies is mAb-825 and mAb-844, as defined below.

One of the antibodies is directed to epitope EP1, as defined below, andthe other antibody binds to epitope EP2, as defined below.

mAb-825:

Rabbit N-terminal mAb-KDM1A (cell Signaling, #2184) that targets anepitope, designated herein EP1, located in the N terminal regionproximate to proline 60 (P60) of the human KDM1A sequence (UNIPROT IDO60341-1) and blocked by peptide #LSD1 Blocking Peptide-2184 specific(Cell Signaling).

mAb-844:

mouse C-terminal mAb-KDM1A (Abcam #ab53269). This antibody targets anepitope, herein designated EP2, located on the C-terminal region andwhich comprises AMYTLPRQATPGVPAQ, corresponding to AA 832-847 of humanKDM1A.

Both antibodies recognize at least human, rat and mouse KDM1A protein.

Further antibodies to KDM1A recognizing epitopes EP1 and EP2 can bereadily developed by methods known in the art. In particular, antibodiesthat bind these epitopes can be identified by their ability to competewith the antibodies described herein or to be blocked by the peptidedescribed above. The sequences comprised in the epitopes EP1 and EP2above correspond to highly conserved regions in the KDM1A protein in thedifferent species, including human, rat and mice.

In some embodiments, in the methods of the invention one of saidantibodies targets epitope EP1, wherein EP1 is located in the N terminalregion proximate to proline 60 (P60) of the human KDM1A sequence(UNIPROT ID 060341-1) and is blocked by peptide #LSD1 BlockingPeptide-2184 specific (Cell Signaling). In some embodiments, the otherof said antibodies targets epitope EP2, wherein EP2 is located on theC-terminal region and comprises AMYTLPRQATPGVPAQ, corresponding to AA832-847 of human KDM1A.

The KDM1A to be used in the herein disclosed test methods and targetengagement assays is preferably a human KDM1A as, inter glia, providedin SEQ ID NO: 1 and the corresponding UNIPROT No. Also KDM1A isoforms ordifferential splice forms and natural or artificial variants of thehuman KDM1A sequence as provided herein and shown in SEQ ID NO:1, inparticular functional homologs, as well as orthologs thereof in otherspecies (e.g. rat, mice, as shown in the appended Examples) may be usedin the test methods and target engagement assays of the invention. TheKDM1A to be contacted with or to be exposed is an expressed protein,which may be naturally expressed, e.g. in a biological sample, or mayalso be recombinantly expressed.

In some embodiments, in the methods of the invention the antibodies aremAb-844 and mAb-825, the tag P in the KDM1A chemoprobe is biotin and theprotein used to capture the chemoprobe is streptavidin.

In some embodiments, in the methods of the invention the level of totalKDM1A is determined by sandwich ELISA and the level of free KDM1A isdetermined by a KDM1A chemoprobe capture ELISA. A schematic depiction ofthis method is shown in FIG. 3. Alternatively, the level of total KDM1Acan be determined by AlphaLISA and the level of free KDM1A can bedetermined by a KDM1A chemoprobe capture AlphaLISA. In some embodiments,the determination of the level of free and total KDM1A is performed ismultiplexed in the same sample.

In some embodiments, the ELISA uses chemoluminescent detection.

It is to be understood that the present invention specifically relatesto each and every combination of features or embodiments describedabove, including any combination of general and/or preferredfeatures/embodiments. In particular, the invention specifically relatesto all combinations of preferred features/embodiments (including alldegrees of preference) of the methods disclosed above.

Other Applications of the Chemoprobes of the Invention

The KDM1A chemoprobes described herein can also be used for the analysisof the spatial distribution of free KDM1A, for example usingchemohistochemistry (CHC), chemofluorescence or flow cytometry onfixed-permeabilized cells or tissues. The concept is similar toimmunohistochemistry (IHC), and comprises contacting a chemoprobe with(a) cell or tissue sample(s), and visualizing the spatial distributionof the chemoprobe through the incorporated tag or label.

Accordingly, the invention provides a method for in vitro determining aspatial distribution of free KDM1A in a sample, wherein said methodcomprises

-   -   (i) contacting or exposing KDM1A to a chemoprobe of the        invention, and    -   (ii) visualizing the spatial distribution of said free KDM1A in        said sample by detection of the chemoprobe.

Detection of KDM1A using a chemoprobe does not only provide informationabout where the protein is, but also on whether the protein is active.

The analysis of the spatial distribution of free KDM1A in a samplethrough detection of the KDM1A bound chemoprobe can be direct, when alabel (as described above) is incorporated into the chemoprobe, orindirect, when a tag (as described above) is incorporated that, in itsturn, can be detected by an agent incorporating a label or any otherentity generating a signal, that can be detected using detection agentsor detection systems, as previously described.

The cells, tissues or organism from which the sample derives may beuntreated or may have been exposed to a KDM1A inhibitor, and in thiscase the KDM1A chemoprobe based method described herein can be used toassess the spatial distribution of free KDM1A, and consequently, thespatial distribution of target engagement. The spatial distribution offree KDM1A using a KDM1A chemoprobe can be assessed in cells sortedusing Fluorescence Assisted Cell Sorting (FACS) with cell type specificmarkers. The cell type specific markers may include cell surface markersknown to be induced by inhibition of KDM1A, p.e. induction of CD11b inTHP-1 AML cells, and inform about the correlation of the degree oftarget inhibition and induction of differentiation at the individualcellular level.

The subcellular localization of free KDM1A can be analyzed using samplessubmitted to fractionation to achieve separation of cell components.

The spatial distribution can also be assessed in samples that conservethe structural integrity of the samples, p.e the KDM1A CHC method can beperformed on tissue sections and in addition it can further be combinedwith traditional IHC, which allows for an assessment of the spatialdistribution of free and total KDM1A protein in (a) cell or tissuesample(s). The KDM1A CHC method can also be combined with IHC directedto other proteins, for example to detect proteins which are expected tobecome up-regulated after KDM1A target engagement, allowing for asimultaneous detection of the target engagement and its down-streameffects.

In addition, the chemoprobes described herein are suitable for theisolation of KDM1A-containing complexes. The invention therefore alsorelates to a method for in vitro determining interaction factors ofKDM1A,

wherein said method comprises

-   -   (i) contacting or exposing a sample to a chemoprobe according to        the invention (e.g. a chemoprobe of the appended claims);    -   (ii) isolating chemoprobe-bound KDM1A-containing complexes;    -   (iii) identifying said interaction factors of KDM1A, wherein        said interaction factors are nucleic acid(s) and/or        polypeptide(s).

To identify the protein partners in KDM1A containing complexes, cell arelyzed in “soft” conditions that don't destroy the protein complexes andcontacted with a KDM1A chemoprobe that contains a tag that can be boundby a capture agent and used to “chemoprecipitate” the KDM1A containingcomplex. The thus obtained “chemoprecipitate” is submitted to proteinanalysis methods known in the art, including the identification ofinteracting proteins by Western blot analysis, microarray analysis usingAntibody-Arrays or mass spectrometry. To minimize false positives,produced by unspecific binding of proteins to the capture agent, anegative control sample can be included that has been exposed to anexcess KDM1A inhibitor, thus blocking the possibility of the chemoprobeto bind the KDM1A complex.

Similarly, the KDM1A chemoprobes described herein can be used to isolateKDM1A target regions in the genome. KDM1A is recruited by transcriptionrepressors to specific sites in the genome (sites vary in different celltypes and under different conditions). The DNA-Protein complexesincluding nucleosomes can be isolated from the nuclei of target cells bycross-linking and digestion of chromatin. DNA associated to KDM1A can beisolated by chemoprecipitation with a KDM1A chemoprobe as describedherein, and selected KDM1A target regions in the genome can beidentified using quantitative PCR (qPCR), or KDM1A target regions can beanalyzed genome wide by Chromatin Chemoprobe precipitation coupled tonext generation sequencing (NGS) or using tiling DNA microarrays. Anadvantage of the KDM1A chemoprobes to the Chromatin Chemoprobeprecipitation technique described herein is the irreversible nature oftheir binding, allowing for stringent washing procedures and highspecificity.

Preparation of the KDM1A Chemoprobes

The chemoprobes of the invention may be made by a variety of methods,including standard chemistry. Illustrative general synthetic methods areset out below and then the preparation of specific compounds of theinvention is described in more detail in the Experimental Section.

As it will be obvious to one skilled in the art, the exact method usedto prepare a given compound may vary depending on its chemicalstructure. Moreover, in some of the processes described below it may benecessary or advisable to protect the reactive or labile groups byconventional protecting groups. Both the nature of these protectinggroups and the procedures for their introduction or removal are wellknown in the art (see for example Greene T. W. and Wuts P. G. M,“Protective Groups in Organic Synthesis”, John Wiley & Sons, 3^(rd)edition, 1999). As an example, as protecting groups of an amino functionthe tert-butoxycarbonyl (Boc) group can be used. Whenever a protectinggroup is present, a later deprotection step will be required, which canbe performed under standard conditions in organic synthesis, such asthose described in the above-mentioned reference. Unless otherwisestated, in the methods described below the meanings of the differentsubstituents are the meanings described above with regard to a compoundof formula I.P-L-Z  I

For instance, compounds of formula I can be prepared by coupling of P toL-Z or alternatively Z to P-L, with both parts convenientlyfunctionalized. Examples of coupling reactions include, but are notlimited to, formation of esters, amides, from activated acids or acylhalides optionally in the presence of a base under standard conditions;formation of sulfonamide by reaction of an amine with a sulfonyl halide,optionally in the presence of a base, under standard conditions;formation of amines by substitution of a primary or secondary amine withan alkylating agent under standard conditions, or by reductiveamination, i.e. by treatment with an aldehyde or a ketone in thepresence of a reducing agent such as sodium cyanoborohydride or sodiumtriacetoxyborohydride; formation of carbamates, ureas or thioureas fromisocyanates, thioisocyanates and chloroformates, under standardconditions; formation of ethers and thioetheres by alkylation ofalcohols and thiols by reaction with alkylating agent under standardconditions.

Examples of reagents that can be used for the attachement of P includeamong others: Alexa Fluor® 405 carboxylic acid, succinimidyl ester;Alexa Fluor® 405 carboxylic acid, succinimidyl ester; Alexa Fluor® 488carboxylic acid, succinimidyl ester; Alexa Fluor® 488 carboxylic acid,2,3,5,6-tetrafluorophenyl ester; Alexa Fluor® 488 5-SDP ester;3-amino-3-deoxydigoxigenin hemisuccinamide, succinimidyl ester;N-(2-aminoethyl)biotinamide, hydrobromide; Alexa Fluor® 405 cadaverine,trisodium salt; Alexa Fluor® 488 cadaverine, sodium salt; Alexa Fluor®555 cadaverine, disodium salt; Alexa Fluor® 568 cadaverine, diammoniumsalt; Alexa Fluor® 594 cadaverine; 5-(aminoacetamido)fluoresceine(fluoresceinyl glycine amide);5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid, sodium salt;7-amino-4-methylcoumarine; 4′-(aminomethyl)fluoresceine, hydrochloride;5-(aminomethyl)fluoresceine, hydrochloride;5-(and-6)-((N-(5-aminopentyl)amino)carbonyl)tetramethylrhodamine;N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide, dipotassiumsalt; 5-((5-aminopentyl)thioureidyl)fluoresceine, dihydrobromide salt;N-(5-aminopentyl)biotinamide, trifluoroacetic acid salt; bimane amine;8-bromomethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene;6-bromoacetyl-2-dimethylaminonaphthalene 5-(bromomethyl)fluoresceine;Biocytine; biocytine hydrazide; 6-((biotinoyl)amino)hexanoic acid,succinimidyl ester; 6-((biotinoyl)amino)hexanoic acid, sulfosuccinimidylester, sodium salt; 6-((6-((biotinoyl)amino)hexanoyl)amino)hexanoicacid, succinimidyl ester;6-((6-((biotinoyl)amino)hexanoyl)amino)hexanoic acid, sulfosuccinimidylester, sodium salt; N-(biotinoyl)-N′-(iodoacetyl)ethylenediamine;D-biotin, succinimidyl ester;5-(((N-(biotinoyl)amino)hexanoyl)amino)pentylamine, trifluoroacetic acidsalt; biotin-X 2,4-dinitrophenyl-X-L-lysine, succinimidyl ester; BODIPY®FL, STP ester, sodium salt; Cascade Blue® acetyl azide, trisodium salt;6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoicacid, succinimidyl ester;6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acid,succinimidyl ester; 6-(2,4-dinitrophenyl)aminohexanoic acid,succinimidyl ester; desthiobiotin-X C2-iodoacetamide;5-dimethylaminonaphthalene-1-(N-(2-aminoethyl))sulfonamide;5-dimethylaminonaphthalene-1-(N-(5-aminopentyl))sulfonamide;6-(fluorescein-5-(and-6)-carboxamido)hexanoic acid, succinimidyl ester;fluorescein-5-EX, succinimidyl ester; lucifer yellow iodoacetamide,dipotassium salt; Lissamine rhodamine B ethylenediamine;Na-(3-maleimidylpropionyl)biocyn; norbiotinamine, hydrochloride;2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate; O6185 OregonGreen® 488-X, succinimidyl ester *6-isomer*; QSY® 7 amine,hydrochloride; Rhodamine Red™-X, succinimidyl ester *5-isomer*;6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid,succinimidyl ester; Texas Red®-X, succinimidyl ester; TS-Link™desthiobiotin-X C5-thiosulfate, sodium salt; Texas Red® cadaverine;

In general, the compounds of formula II can be obtained in two steps bythe method described in Scheme 1:

wherein P, L₁, X₁, X₂, Z₁, Z₂, R₅, R₆ and D have the meaning previouslydescribed in relation with a compound of formula II; PG represents anamine protecting group, such as for example tert-butoxycarbonyl (Boc);and X_(1a) and X_(1b) represent functional groups as —C(═O)OH, —C(═O)Cl,-halogen, —C(═O)H, —NH₂, —NHR₁, —N═C═O, —N═C═S, —OH, —SO₂Cl, —SH,—O(C═O)Cl, that by appropriate conversion reactions can generate X₁.

In a first step (step a), a compound of formula II-A can be prepared bycoupling of compound of formula II-B to with both parts bearingconvenient reactive functions as X_(1a) and X_(1b) than can generetate

For example, when X₁ is —NR₁—C(═O)— or —C(═O)—NR₁—, X_(1a) is acarboxylic acid (—C(═O)OH) and X_(1b) is an amino group (—NHR₁) and viceversa, compounds of formula II-A can be obtained by means of activatingagents. Examples of said activating agents are among others:dicyclohexyl carbodiimide (DCC), 1-hydroxybenzotriazole (HOBT),N-hydroxysuccinimide (HOSu), 1-ethyl-3-(3′-dimethylamino)carbodiimide(EDC), diisopropyl carbodiimide (DIC), carbonyl diimidazole (CDI),Benzotriazol-1-yl-oxytris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP),Benzotriazol-1-yl-oxytris-pyrrolidinophosphonium hexafluorophosphate(PyBop), O-(1Hbenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU),1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU). Reaction can be carried out in thepresence of a base, such as, disopropylethylamine, pyridine,thriethylamine, or N-methylmorpholine, in a solvent, such asdimethoxyethane, N,N-dimethylformamide, tetrahydrofuran, dichloromethaneor dioxane. Alternatively, carboxylic acids (C(═O)OH) are activated asmixed anhydrides or acid chlorides and then coupled with amides (—NHR₁)in the presence of a suitable base such as sodium hydride,triethylamine, diisopropylethylamine, pyridine or the like.

For example, when X₁ is —NR₁—, X_(1a) is a ketone or an aldehyde (—C(═O)or —C(═O)H) and X_(1b) is an amino group (—NHR₁) and vice versa,compounds of formula II-A can be obtained by means of reductiveamination or alkylation in the presence of a reducing agent such assodium cyanoborohydride or sodium thacetoxyborohydride in a suitablesolvent such as dioxane, THF, dichloromethane or diethyl ether.Alternatively, when X₁ is —NR₁—, X₁, is a alkyl halide (—X) and X_(1b)is an amino group (—NHR₁) and vice versa, compounds of formula II-A canbe obtained by means of amine alkylation in the presence of a bases suchas sodium carbonate, potassium carbonate, cesium carbonate, potassiumphosphate or the like. Other preferred solvents include dioxane,tetrahydrofuran and pyridine. Temperature can be varied from roomtemperature to 100° C.

For example, when X₁ is —SO₂—NR₁— or —SO₂—NR₁—, X_(1a) is a sulfonylchloride (—SO₂Cl)—) and X_(1b) is an amino group (—NHR₁) and vice versa,compounds of formula II-A can be obtained by means of direct reaction ofboth compounds optionally in the presence of a base such as4-dimethylaminopyhdine, in a suitable solvent such as dioxane,chloroform, dichloromethane or pyridine.

For example, when X₁ is —O—C(═O)—NR¹— or —NR¹—C(═O)—O—, X_(1a) is achloroformate (—O(C═O)Cl) and X_(1b) is an amino group (—NHR1) and viceversa, compounds of formula II-A can be obtained by means of directreaction of both compounds optionally in the presence of a base such as4-dimethylaminopyhdine, in a suitable solvent such as dioxane,chloroform, dichloromethane or pyridine.

For example, when X₁ is —C(═O)—O— or —O—C(═O)—, X_(1a) is a carboxylicacid (—(C═O)—OH) and X_(1b) is an alcohol group (—OH) and vice versa,compounds of formula II-A can be obtained by means of activating agents.Examples of said activating agents are among others: dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBT), N-hydroxysuccinimide(HOSu), 1-ethyl-3-(3′-dimethylamino)carbodiimide (EDC), diisopropylcarbodiimide (DIC), carbonyl diimidazole (CDI),Benzotriazol-1-yl-oxytris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP),Benzotriazol-1-yl-oxytris-pyrrolidinophosphonium hexafluorophosphate(PyBop), O-(1Hbenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU),1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU). Reaction can be carried out in thepresence of a base, such as, disopropylethylamine, pyridine,thriethylamine, or N-methylmorpholine, in a solvent, such asdimethoxyethane, N,N-dimethylformamide, tetrahydrofuran, dichloromethaneor dioxane. Alternatively, when X₁ is —C(═O)—O— or —O—C(═O)—, X_(1a) isa carboxylic acid (—(C═O)—OH) and X_(1b) is an alcohol group (—OH) andvice versa, compounds of formula II-A can be obtained by Fisheresterification of both compounds in the presence of catalytic amounts ofan acid as sulfuric acid, thionyl chloride, TMSCI, pTSOH, HCl, oroxallyl chloride.

For example, when X₁ is —O—, X_(1a) is a alkyl halide (—X) and X_(1b) isan alcohol group (—OH) and vice versa, compounds of formula II-A can beobtained by Williamson ether synthesis in presence of a base such ussodium hydride, potassium tert-butoxide, sodium tert-butoxide, sodiumhydroxide, potassium hydroxide, or a phase transfer catalyst such usbenzyltrimethylammonium chloride, benzyltriethylammonium chloride,methyltricaprylammonium chloride, methyltributylammonium chloride,methyltrioctylammonium chloride, or hexadecyltributylphosphoniumbromide.

For example, when X₁ is —NR1-C(═O)—NR1-, X_(1a) is a alkyl isocyanate(—N═C═O) and X_(1b) is an amino group (—NHR₁) and vice versa, compoundsof formula II-A can be obtained by means of direct reaction of bothcompounds, in a suitable solvent such as dioxane, toluene, orN,N-dimethylformamide.

For example, when X₁ is —NR1-C(═S)—NR1-, X_(1a) is a alkyl isotiocyanate(—N═C═S) and X_(1b) is an amino group (—NHR₁) and vice versa, compoundsof formula II-A can be obtained by means of direct reaction of bothcompounds, in a suitable solvent such as dioxane, toluene, orN,N-dimethylformamide.

In the last step (step b), the protecting group of a compound of formulaII-A is cleavaged under the standard conditions described in theliterature to give a compound II. For example, in case Boc is used asPG, the cleavage is typically performed by treating compound II-A with a4M dioxane/HCl(_(g)) mixture at room temperature

A compound of formula II-B can be obtained by reaction of a compound offormula II-E with a compound of formula II-D, as shown in Scheme 2,under the conditions reported in the literature for Suzuki couplings.For example, the reaction can be carried out in the presence of a base,such as Na₂CO₃, NaOH, Cs₂CO₃, CsF or Ba(OH)₂, and a palladium catalyst,such as Pd(PPh₃)₄, Pd₂(dba)₃ or Pd(OAc)₂, in a solvent, such asdimethoxyethane, toluene, N,N-dimethylformamide, tetrahydrofuran ordioxane, optionally in the presence of water, and heating, preferably ataround 90° C.

The compound of formula II-D can be prepared in two steps, as shown inScheme 3. In a first step (step a), a compound of formula II-G isreacted with a commercially available aldehyde or ketone under reductivealkylation standard conditions, for example using sodiumtriacetoxyborohydride or sodium borohydride as reducing agent in asuitable solvent such as dichloroethane or methanol leading to theformation of (trans)-cyclopropylamino derivatives of formula II-F. In asecond step, the amino group is protected with a Boc protecting group(Boc: tert-butoxycarbonyl), under standard conditions, for example usingBoc anhydride and a base, preferably triethylamine in a suitable solventsuch as THF or ACN to give a compound of formula II-D.

Alternatively, compounds of formula II can be prepared by coupling of Pto L₁ in the final steps with both parts conveniently functionalized.Examples of coupling reactions include, but are not limited to,formation of —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹—, —NR¹—SO₂— or —O— groups. The synthetic methods described forthe preparation of X₁ groups in compounds of formula II can be appliedto coupling of P to L₁.

Compounds of formula IIa, III and IIIa can be prepared likewise.

Furthermore, some compounds of the present invention can also beobtained from other compounds of formula I by appropriate conversionreactions of functional groups in one or several steps, using well-knownreactions in organic chemistry under the standard experimentalconditions.

Said transformations include, for example: the reduction of a nitrogroup to give an amino group, for example by treatment with hydrogen,hydrazine or formic acid in the presence of a suitable catalyst such asPd/C; or by treatment with sodium borohydride in the presence of NiCl₂,Or SnCl₂; the substitution of a primary or secondary amine by treatmentwith an alkylating agent under standard conditions, or by reductiveamination, i.e. by treatment with an aldehyde or a ketone in thepresence of a reducing agent such as sodium cyanoborohydride or sodiumthacetoxyborohydride; the conversion of an amine into a sulfonamide byreaction with a sulfonyl halide, such as sulfonyl chloride, optionallyin the presence of a base such as 4-dimethylaminopyhdine, in a suitablesolvent such as dioxane, chloroform, dichloromethane or pyridine,optionally in the presence of a base such as triethylamine or pyridine;the conversion of an amine into an amide, carbamate or urea understandard conditions; the alkylation of an amide by treatment with analkylating agent under basic conditions; the conversion of an alcoholinto an ether, ester or carbamate under standard conditions; thealkylation of a thiol to give a thioeter under standard conditions; thepartial or total oxidation of an alcohol to give ketones, aldehydes orcarboxylic acids under standard oxidizing conditions; the reduction ofan aldehyde or ketone by treatment with a reducing agent such as sodiumborohydride; the reduction of a carboxylic acid or a carboxylic acidderivative to an alcohol by treatment with a reducing agent such asdiisobutylaluminium hydride or LiAlH₄; the oxidation of a thioeter to asulfoxide or sulfone under standard conditions; the conversion of analcohol into a halogen by reaction with SOCl₂, PBr₃, tetrabutylammoniumbromide in the presence of P₂O₅, or PCl₃; the conversion of halogen intoan amine by reaction with an amine, optionally in the presence of asuitable solvent, and preferably heating; and the conversion of aprimary amide into a —CN group under standard conditions.

Likewise, any of the aromatic rings of the compounds of the presentinvention can undergo electrophilic aromatic substitution reactions ornucleophilic aromatic substitution reactions, widely described in theliterature. Some of these interconversion reactions are explained ingreater detail in the examples. As it will be obvious to those skilledin the art, these interconversion reactions can be carried out upon thecompounds of formula I as well as upon any suitable synthesisintermediate thereof.

Any reference in the synthetic methods described above to a compound offormula I apply likewise to a compound of formula II, IIa, III and IIIa.

KDM1A Inhibitors

The methods, assays and chemoprobes of the invention can be used todetermine target engagement of KDM1A inhibitors.

Both irreversible and reversible KDM1A inhibitors have been reported.Irreversible KDM1A inhibitors exert their inhibitory activity bybecoming covalently bound to the FAD cofactor within the KDM1A activesite and are generally based on a 2-cyclyl-cyclopropylamino moiety suchas a 2-(hetero)arylcyclopropylamino moiety. Reversible inhibitors ofKDM1A have also been disclosed.

Non-limiting examples of KDM1A inhibitors are disclosed e.g. in:WO2010/043721, WO2010/084160, WO2011/035941, WO2011/042217,WO2011/131697, WO2012/013727, WO2012/013728, WO2012/045883,WO2013/057320, WO2013/057322, WO2010/143582, US2010-0324147,WO2011/022489, WO2011/131576, WO2012/034116, WO2012/135113,WO2013/022047, WO2013/025805, WO2014/058071, WO2014/084298,WO2014/086790, WO2014/164867, WO2014/205213, WO2015/021128,WO2015/031564, US2015-0065434, WO2007/021839, WO2008/127734,WO2015/089192, CN104119280, 0N103961340, CN103893163, CN103319466,ON103054869, WO2015/123408, WO2015/123424, WO2015/123437, WO2015/123465,WO2015/156417, WO2015/181380, WO2016/123387, WO2016/130952,WO2016/172496, WO2016/177656, WO2017/027678, CN106045862, WO2012/071469,WO2013/033688, WO2014/085613, WO2015/120281, WO2015/134973,WO2015/168466, WO2015/200843, WO2016/003917, WO2016/004105,WO2016/007722, WO2016/007727, WO20161007731, WO2016/007736,WO2016/034946, WO2016/037005, WO2016/161282, and WO2017/004519 K Taekoet al, Bioorg Med Chem Lett 2015, 25(9):1925-8. doi:10.1016/j.bmc1.2015.03.030. Epub 2015 Mar. 20, PMID: 25827526; S Valenteet al, Eur J Med Chem. 2015, 94:163-74. dol:10.1016/j.ejmech.2015.02.060. Epub 2015 Mar. 3, PMID:25768700; MN AhmedKhan et al Med. Chem. Commun., 2015, 6, 407-412, DOI: 10.1039/C4MD00330Fepub 29 Sep. 2014; M Pieroni et al, Eur J Med Chem. 2015; 92:377-386.doi: 10.1016/j.ejmech.2014.12.032. Epub 2015 Jan. 7. PMID:25585008; VRodriguez et al, Med. Chem. Commun., 2015, 6, 665-670 DOI:10.1039/C4MD00507D, Epub 23 Dec. 2014; P Vianello et al, Eur J Med Chem.2014, 86:352-63. doi: 10.1016/j.ejmech.2014.08.068. Epub 2014 Aug. 27;DP Mould et al, Med. Res. Rev., 2015, 35:586-618. doi:10.1002/med.21334,epub 24 Nov. 2014; L Y Ma et al, 2015, 58(4):1705-16. doi:10.1021/acs.jmedchem.5b00037. Epub 2015 Feb. 6; S L Nowotarski et al,2015, 23(7):1601-12. doi: 10.1016/j.bmc.2015.01.049. Epub 2015 Feb. 7.PMID:25725609; C J Kutz et al Medchemcomm. 2014, 5(12):1863-1870 PMID:25580204; C Zhou et al, Chemical Biology & Drug Design, 2015,85(6):659-671. doi:10.1111/cbdd.12461, epub 22 Dec. 2014; P Prusevich etal, ACS Chem Biol. 2014, 9(6):1284-93. doi: 10.1021/cb500018s. Epub 2014Apr. 7; B Dulla et al, Org Biomol Chem 2013, 11, 3103-3107, doi:10.1039/c3ob40217g; J R Hitchin et al, MedChemCommun, 2013, 4, 1513-1522DOI: 10.1039/c3md00226h; and Y Zhou et al, Biorg Med Chem Lett, 2015,online publication 20 Jun. 2015, doi:10.1016/j.bmc1.2015.06.054.

Irreversible KDM1A inhibitors (which are nonpeptidic) that can bestudied using the chemoprobes and methods of the invention include,without limitation, the compounds disclosed in: WO2010/043721,WO2010/084160, WO2011/035941, WO2011/042217, WO2011/131697,WO2012/013727, WO2012/013728, WO2012/045883, WO2013/057320,WO2013/057322, WO2010/143582, US2010-0324147, WO2011/131576,WO2012/135113, WO2013/022047, WO2014/058071, WO2014/084298,WO2014/086790, WO2014/164867, WO2015/021128; WO2015/123408,WO2015/123424, WO2015/123437, WO2015/123465, WO2015/156417,WO2015/181380, WO2016/123387, WO2016/130952, WO2016/172496,WO2016/177656, WO2017/027678, CN106045862, K Taeko et al, Bioorg MedChem Lett. 2015, 25(9):1925-8. doi: 10.1016/j.bmc1.2015.03.030. Epub2015 Mar. 20, PMID: 25827526; S Valente et al, Eur J Med Chem. 2015,94:163-74. doi: 10.1016/j.ejmech.2015.02.060. Epub 2015 Mar. 3,PMID:25768700; M N Ahmed Khan et al Med. Chem. Commun., 2015, 6,407-412, DOI: 10.1039/C4MD00330F epub 29 Sep. 2014; M Pieroni et al, EurJ Med Chem. 2015; 92:377-386. doi: 10.1016/j.ejmech.2014.12.032. Epub2015 Jan. 7. PMID:25585008; V Rodriguez et al, Med. Chem. Commun., 2015,6, 665-670 DOI: 10.1039/C4MD00507D, Epub 23 Dec. 2014; P Vianello et al,Eur J Med Chem. 2014, 86:352-63. doi: 10.1016/j.ejmech.2014.08.068. Epub2014 Aug. 27.

Reversible KDM1A inhibitors (which are nonpeptidic) that can be studiedusing the chemoprobes and methods of the invention include, withoutlimitation, the compounds disclosed in WO2007/021839, WO2008/127734,WO2011/022489, WO2012/034116, WO2012/071469, WO2013/025805,US201510065434, WO2013/033688, CN103054869, CN103319466, WO2014/085613,CN103893163A, CN103961340, WO2014/205213, WO2015/031564, WO2015/089192,WO2015/120281, WO2015/134973, WO2015/168466, WO2015/200843,WO2016/003917, WO2016/004105, WO2016/007722, WO2016/007727,WO2016/007731, WO2016/007736, WO2016/034946, WO20161037005,WO2016/161282, and WO2017/004519.

In the methods and uses according to the invention, the KDM1A inhibitoris preferably an irreversible KDM1A inhibitor, preferably a2-(hetero)arylcyclopropylamino KDM1A inhibitor. As used herein, a“2-(hetero)arylcyclopropylamino KDM1A inhibitor” or a“2-(hetero)arylcyclopropylamino compound” means a KDM1A inhibitor whosechemical structure comprises a cyclopropyl ring substituted at position1 with an amino group, which is optionally substituted, and substitutedat position 2 with an aryl or heteroaryl group (wherein the aryl orheteroaryl group is optionally substituted).

The ability of a compound to inhibit KDM1A can be tested in vitro usingany method to determine KDM1A inhibition known in the art, for examplethe method disclosed in Example 2.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (A) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

In formula (I), each of R1-R5 is optionally substituted andindependently chosen from —H, halo, alkyl, alkoxy, cycloalkoxy,haloalkyl, haloalkoxy, -L-aryl, -L-heteroaryl, -L-heterocyclyl,-L-carbocycle, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl,amino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy,arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl,heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro,sulfinyl, sulfonyl, sulfonamide, thiocarbonyl, thiocyanato,trihalomethanesulfonamido, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, and C-amido;

R6 is chosen from —H and alkyl;

R7 is chosen from —H, alkyl, and cycloalkyl;

R8 is chosen from —C(═O)NR_(x)R_(y) and —C(═O)R_(z);

R_(x) when present is chosen from —H, alkyl, alkynyl, alkenyl,-L-carbocycle, -L-aryl, -L-heterocyclyl, all of which are optionallysubstituted;

R_(y) when present is chosen from —H, alkyl, alkynyl, alkenyl,-L-carbocycle, -L-aryl, -L-heterocyclyl, all of which are optionallysubstituted;

R_(z) when present is chosen from —H, alkoxy, -L-carbocyclic,-L-heterocyclic, -L-aryl, wherein the aryl, heterocyclyl, or carbocycleis optionally substituted;

each L can be saturated, partially saturated, or unsaturated, and isindependently chosen from —(CH₂)_(n)—(CH₂)_(n)—,—(CH₂)_(n)C(═O)(CH₂)_(n)—, —(CH₂)_(n)C(═O)NH(CH₂)_(n)—,—(CH₂)_(n)NHC(═O)O(CH₂)_(n)—, —(CH₂)_(n)NHC(═O)NH(CH₂)_(n)—,—(CH₂)_(n)NHC(═S)S(CH₂)_(n)—, —(CH₂)_(n)OC(═O)S(CH₂)_(n)—,—(CH₂)_(n)NH(CH₂)_(n)—, —(CH₂)_(n)O(CH₂)_(n)—, —(CH₂)_(n)S(CH₂)_(n)—,and —(CH₂)_(n)NHC(═S)NH(CH₂)_(n)—, where each n is independently chosenfrom 0, 1, 2, 3, 4, 5, 6, 7, and 8, wherein optionally substitutedrefers to zero or 1 to 4 optional substituents independently chosen fromacylamino, acyloxy, alkenyl, alkoxy, cycloalkoxy, alkyl, alkylthio,cycloalkylthio, alkynyl, amino, aryl, arylalkyl, arylalkenyl,arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, carbocyclyl,cyano, cyanato, halo, haloalkyl, haloaryl, hydroxyl, heteroaryl,heteroaryloxy, heterocyclyl, heteroarylalkoxy, isocyanato,isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl,thiocyanato, trihalomethanesulfonamido, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, and C-amido.

Compounds of formula (A) having a (trans) disposition on thesubstituents on the cyclopropyl ring are preferred.

Preferably, the compound of formula (A) is a compound from the listbelow:

-   N-cyclopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;-   2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;-   N-cyclopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}propanamide;-   2-{[(trans)-2-phenylcyclopropyl]amino}-N-prop-2-ynylacetamide;-   N-isopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;-   N-(tert-butyl)-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;-   N-(2-morpholin-4-yl-2-oxoethyl)-N-[(trans)-2-phenylcyclopropyl]amine;-   2-{[(trans)-2-phenylcyclopropyl]amino}propanannide;-   Methyl 2-{[(trans)-2-phenylcyclopropyl]amino}propanoate;-   N-cyclopropyl-2-{methyl[(trans)-2-phenylcyclopropyl]amino}acetamide;-   2-{methyl[(trans)-2-phenylcyclopropyl]amino}acetamide;-   N-methyl-trans-2-(Phenylcyclopropylamino)propanamide;-   1-(4-methylpiperazin-1-yl)-2-((trans)-2-phenylcyclopropylamino)ethanone;-   1-(4-ethylpiperazin-1-yl)-2-((trans)-2-phenylcyclopropylamino)ethanone;-   1-(4-benzylpiperazin-1-yl)-2-((trans)-2-phenylcyclopropylamino)ethanone;-   2-((trans)-2-phenylcyclopropylamino)-1-(4-phenylpiperazin-1-yl)ethanone;-   2-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;-   2-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylannino)-N-cyclopropylacetamide;-   2-((trans)-2-(4-(3-fluorobenzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;-   2-((trans)-2-(4-(3-chlorobenzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;-   2-((trans)-2-(biphenyl-4-yl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;-   1-(4-methylpiperazin-1-yl)-2-((trans)-2-(4-phenethoxyphenyl)cyclopropylamino)ethanone;-   2-((trans)-2-(4-(4-fluorobenzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;-   2-((trans)-2-(4-(biphenyl-4-ylmethoxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;-   2-({(trans)-2-[4-(benzyloxy)phenyl]cyclopropyl}amino)-N-cyclopropylacetamide,-   N-[(trans)-2-(4-benzyloxyphenyl)cyclopropyl]}-N-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]amine,-   N-[2-oxo-2-(4-phenylpiperazin-1-yl)ethyl]-N-[(trans)-2-phenylcyclopropyl]amine,-   N-[2-(4-benzylpiperazin-1-yl)-2-oxoethyl]-N-[(trans)-2-phenylcyclopropyl]amine,-   N-[2-(4-ethylpiperazin-1-yl)-2-oxoethyl]-N-[(trans)-2-phenylcyclopropyl]amine,-   N-[2-(4-methylpiperazin-1-yl)-2-oxoethyl-]-N-[(trans)-2-phenylcyclopropyl]amine,-   2-((trans)-2-(4-pyridin-3-ylphenyl)    cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone, and-   2-((trans)-2-(3′-methoxy-1,1′-biphenyl-4-yl)    cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone,

and pharmaceutically acceptable salts thereof.

Compounds of formula (A) can be prepared by the methods disclosed inWO2010/043721, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (B) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

In formula (B), each of R1-R5 is independently chosen from —H, halo,alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl,-L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio,cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl,arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio,cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy,isocyanato, isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamido,thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, and C-amido;

R6 is chosen from —H and alkyl;

R7 is chosen from —H, alkyl, and cycloalkyl;

R8 is a -L-heterocyclyl wherein the ring or ring system of said-L-heterocyclyl has from 0-3 substituents chosen from halo, alkyl,alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl,-L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl,amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl,arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl,hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanato,nitro, sulfinyl, sulfonyl, sulfonamido, thiocarbonyl, thiocyanato,trihalomethanesulfonamido, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, and C-amido; or

R8 is -L-aryl wherein the ring or ring system of said -L-aryl has from1-3 substituents chosen from halo, alkyl, alkoxy, cycloalkoxy,haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl,acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino,alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy,aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl,heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro,sulfinyl, sulfonyl, sulfonamido, thiocarbonyl, thiocyanato,trihalomethanesulfonamido, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, and C-amido;

each L is independently chosen from —(CH₂)_(n)—(CH₂)_(n)—,—(CH₂)_(n)NH(CH₂)_(n)—, —(CH₂)_(n)O(CH₂)_(n)—, and—(CH₂)_(n)S(CH₂)_(n)—, and where each n is independently chosen from 0,1, 2, and 3.

Compounds of formula (B) having a (trans) disposition on thesubstituents on the cyclopropyl ring are preferred.

Preferably the compound of formula (B) is a compound from the listbelow:

-   (trans)-N-(4-fluorobenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(4-fluorobenzyl)-2-phenylcyclopropanaminium;-   4-(((trans)-2-phenylcyclopropylamino)methyl)benzonitrile;-   (trans)-N-(4-cyanobenzyl)-2-phenylcyclopropanaminium;-   (trans)-2-phenyl-N-(4-(trifluoromethyl)benzyl)cyclopropanamine;-   (trans)-2-phenyl-N-(4-(trifluoromethyl)benzyl)cyclopropanaminium;-   (trans)-2-phenyl-N-(pyridin-2-ylmethyl)cyclopropanamine;-   (trans)-2-phenyl-N-(pyridin-3-ylmethyl)cyclopropanamine;-   (trans)-2-phenyl-N-(pyridin-4-ylmethyl)cyclopropanamine;-   (trans)-N-((6-methylpyridin-2-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-2-phenyl-N-(thiazol-2-ylmethyl)cyclopropanamine;-   (trans)-2-phenyl-N-(thiophen-2-ylmethyl)cyclopropanamine;-   (trans)-N-((3-bromothiophen-2-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N4-((4-bromothiophen-2-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-(3,4-dichlorobenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(3-fluorobenzyl)-2-phenylcyclopropanaminium;-   (trans)-N-(2-fluorobenzyl)-2-phenylcyclopropanamine;-   (trans)-2-phenyl-N-(quinolin-4-ylmethyl)cyclopropanamine;-   (trans)-N-(3-methoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-2-phenyl-N-((6-(trifluoromethyl)pyridin-3-yl)methyl)cyclopropanamine;-   (trans)-N-((6-chloropyridin-3-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-((4-methylpyridin-2-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N4-((6-methoxypyridin-2-yl)methyl)-2-phenylcyclopropanamine;-   2-(((trans)-2-phenylcyclopropylamino)methyl)pyridin-3-ol;-   (trans)-N-((6-bromopyridin-2-yl)methyl)-2-phenylcyclopropanamine;-   4-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)benzonitrile;-   (trans)-N-(4-(benzyloxy)benzyl)-2-phenylcyclopropanamine;-   (trans)-N-benzyl-2-(4-(benzyloxy)phenyl)cyclopropanamine;-   (trans)-2-(4-(benzyloxy)phenyl)-N-(4-methoxybenzyl)cyclopropanamine;-   (trans)-2-(4-(benzyloxy)phenyl)-N-(4-fluorobenzyl)cyclopropanamine;-   (trans)-2-phenyl-N-(quinolin-2-ylmethyl)cyclopropanamine;-   (trans)-2-phenyl-N-((5-(trifluoromethyl)pyridin-2-yl)methyl)cyclopropanamine;-   (trans)-N-((3-fluoropyridin-2-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-2-phenyl-N-(quinolin-3-ylmethyl)cyclopropanamine;-   (trans)-N4-((6-methoxypyridin-3-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N4-((5-methoxypyridin-3-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-((3H-indol-3-yl)methyl)-2-phenylcyclopropanamine;-   3-(((trans)-2-phenylcyclopropylamino)methyl)benzonitrile;-   (trans)-N-(2-methoxybenzyl)-2-phenylcyclopropanamine;-   3-(((trans)-2-phenylcyclopropylamino)methyl)pyridin-2-amine;-   (trans)-N-((2-chloropyridin-3-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-(3,4-dimethoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-phenylcyclopropanamine;-   (trans)-N-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-(2,6-difluoro-4-methoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-2-phenyl-N-(4-(trifluoromethoxy)benzyl)cyclopropanamine;-   (trans)-N-(5-fluoro-2-methoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(2-fluoro-4-methoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-((4-methoxynaphthalen-1-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-(2-fluoro-6-methoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-((2-methoxynaphthalen-1-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-((4,7-dimethoxynaphthalen-1-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-(4-methoxy-3-methylbenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(3-chloro-4-methoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(3-fluoro-4-methoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(4-methoxy-2-methylbenzyl)-2-phenylcyclopropanamine;-   (trans)-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-6-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-((2,2-dimethylchroman-6-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-N-(4-methoxy-2,3-dimethylbenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(4-methoxy-2,5-dimethylbenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(2-fluoro-4,5-dimethoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(3-chloro-4,5-dimethoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(2-chloro-3,4-dimethoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(2,4-dimethoxy-6-methylbenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(2,5-dimethoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(2,3-dimethoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-(2-chloro-3-methoxybenzyl)-2-phenylcyclopropanamine;-   (trans)-N-((1H-indol-5-yl)methyl)-2-phenylcyclopropanamine;-   (trans)-2-(4-(benzyloxy)phenyl)-N-(pyridin-2-ylmethyl)cyclopropanamine;-   (trans)-2-(4-(benzyloxy)phenyl)-N-(2-methoxybenzyl)cyclopropanamine;-   (trans)-N-(1-(4-methoxyphenyl)ethyl)-2-phenylcyclopropanamine;-   (trans)-N-(1-(3,4-dimethoxyphenyl)ethyl)-2-phenylcyclopropanamine;-   (trans)-N-(1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethyl)-2-phenylcyclopropanamine;-   (trans)-N-(1-(5-fluoro-2-methoxyphenyl)ethyl)-2-phenylcyclopropanamine;-   (trans)-N-(1-(3,4-dimethoxyphenyl)propan-2-yl)-2-phenylcyclopropanamine;-   (trans)-N-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)-2-phenylcyclopropanamine;    and

pharmaceutically acceptable salts thereof.

Compounds of formula (B) can be prepared by the methods disclosed inWO2010/084160, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (C) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:(A′)_(x)-(A)-(B)—(Z)-(L)-(D)  (C)

In formula (C), (A) is heteroaryl or aryl;

each (A′), if present, is independently chosen from aryl, arylalkoxy,arylalkyl, heterocyclyl, aryloxy, halo, alkoxy, haloalkyl, cycloalkyl,haloalkoxy, and cyano, wherein each (A′) is substituted with 0, 1, 2, or3 substituents independently chosen from halo, haloalkyl, aryl,arylalkoxy, alkyl, alkoxy, cyano, sulfonyl, amido, and sulfinyl;

X is 0, 1, 2, or 3;

(B) is a cyclopropyl ring, wherein (A) and (Z) are covalently bonded todifferent carbon atoms of (B);

(Z) is —NH—;

(L) is chosen from —CH₂CH₂—, —CH₂CH₂CH₂—, and —CH₂CH₂CH₂CH₂—; and

(D) is chosen from —N(—R1)-R2, —O—R3, and —S—R3, wherein:

R1 and R2 are mutually linked to form a heterocyclic ring together withthe nitrogen atom that R1 and R2 are attached to, wherein saidheterocyclic ring has 0, 1, 2, or 3 substituents independently chosenfrom —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₅ alkyl)(C₁-C₆ alkyl), alkyl, halo,cyano, alkoxy, haloalkyl, and haloalkoxy, or

R1 and R2 are independently chosen from —H, alkyl, cycloalkyl,haloalkyl, and heterocyclyl, wherein the sum of substituents on R1 andR2 together is 0, 1, 2, or 3, and the substituents are independentlychosen from —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)(C₁-C₆ alkyl), andfluoro; and

R3 is chosen from —H, alkyl, cycloalkyl, haloalkyl, and heterocyclyl,wherein R3 has 0, 1, 2, or 3 substituents independently chosen from—NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)(C₁-C₆ alkyl), and fluoro.

Compounds of formula (C) having a (trans) disposition on thesubstituents on the cyclopropyl ring are preferred.

Preferably the compound of formula (C) is a compound from the listbelow:

-   N-[2-(4-methylpiperazin-1-yl)ethyl]-N-[(trans)-2-phenylcyclopropyl]    amine;-   N-cyclopropyl-N′-[(trans)-2-phenylcyclopropyl]ethane-1,2-diamine;-   N,N-dimethyl-N′-(2-{[(trans)-2-phenylcyclopropyl]amino}ethyl)ethane-1,2-diamine;-   (3R)-1-(2-{[(trans)-2-phenylcyclopropyl]amino}ethyl)pyrrolidin-3-amine;-   (3S)—N,N-dimethyl-1-(2-{[(trans)-2-phenylcyclopropyl]amino}ethyl)    pyrrolidin-3-amine;-   (3R)—N,N-dimethyl-1-(2-{[(trans)-2-phenylcyclopropyl]amino}ethyl)pyrrolidin-3-amine;-   N-[(trans)-2-phenylcyclopropyl]-N-(2-piperazin-1-ylethyl)amine;-   N1,N1-diethyl-N2-((trans)-2-phenylcyclopropyl)ethane-1,2-diamine;-   N-[(trans)-2-phenylcyclopropyl]-N-(2-piperidin-1-ylethyl)amine;-   (trans)-2-(4-(benzyloxy)phenyl)-N-(2-(4-methylpiperazin-1-yl)ethyl)    cyclopropanamine;-   (trans)-N-(2-(4-methylpiperazin-1-yl)ethyl)-2-(3′-(trifluoromethyl)    biphenyl-4-yl)cyclopropanamine;-   (trans)-2-(3′-chlorobiphenyl-4-yl)-N-(2-(4-methylpiperazin-1-yl)ethyl)    cyclopropanamine;-   (R)-1-(2-((trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;    and-   N¹-cyclopropyl-N²-((trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl)    cyclopropyl)ethane-1,2-diamine;-   N1-((trans)-2-(4-(3-bromobenzyloxy)phenyl)cyclopropyl)-N2-cyclopropylethane-1,2-diamine;-   N1-((trans)-2-(3′-chlorobiphenyl-4-yl)cyclopropyl)-N2-cyclopropylethane-1,2-diamine;-   N1-cyclopropyl-N2-((trans)-2-(4-phenethoxyphenyl)cyclopropyl)ethane-1,2-diamine;-   N1,N1-diethyl-N2-((trans)-2-(4-(3-fluorobenzyloxy)phenyl)cyclopropyl)ethane-1,2-diamine;-   (trans)-2-(4-bromophenyl)-N-(2-(4-methylpiperazin-1-yl)ethyl)cyclopropanamine;-   N1-((trans)-2-(terphenyl-4-yl)cyclopropyl)-N2-cyclopropylethane-1,2-diamine;-   (trans)-N-(2-(piperidin-1-yl)ethyl)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropanamine;-   N1,N1-diethyl-N2-((trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropyl)ethane-1,2-diamine;-   (trans)-N-(2-(piperazin-1-yl)ethyl)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropanamine;-   (S)-1-(2-((trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(3′-chlorobiphenyl-4-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(4′-chlorobiphenyl-4-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(3′-methoxybiphenyl-4-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(4-(3-bromobenzyloxy)phenyl)cyclopropylamino)ethyl)pyrrolidin-3-amine;    and-   (R)-1-(2-((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   N-(trans)-2-(isobutylthio)-ethyl-2-phenylcyclopropanamine,-   N-trans-(2-ethoxyethyl)-2-phenylcyclopropanamine, and-   N-trans-(2-methoxyethyl)-2-phenylcyclopropanamine,-   (R)-1-(2-((trans)-2-(4-(4-bromobenzyloxy)phenyl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(4-(4-chlorobenzyloxy)phenyl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(4-(biphenyl-4-ylmethoxy)phenyl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(3′,5′-dichlorobiphenyl-4-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   N1-((trans)2-(2-[1,1′;4′,1″]terphenyl-4″-yl-cyclopropyl)-N2-cyclopropylethane-1,2-diamine;-   (R)-1-(2-((trans)-2-(6-(benzyloxy)-4′-(trifluoromethyl)biphenyl-3-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;    and-   (R)-1-(2-((trans)-2-(6-(benzyloxy)biphenyl-3-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(4-phenethoxyphenyl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(6-(3-methoxyphenyl)pyridin-3-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;-   (R)-1-(2-((trans)-2-(6-(4-chlorophenyl)pyridin-3-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;    and-   4-((4-((trans)-2-(2-((R)-3-aminopyrrolidin-1-yl)ethylamino)cyclopropyl)phenoxy)methyl)benzonitrile;

and pharmaceutically acceptable salts thereof.

Compounds of formula (C) can be prepared by the methods disclosed inWO2011/035941, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (D) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:(A′)_(x)-(A)-(B)—(Z)-(L)-C(═O)NH₂  (D)

In formula (D), (A) is heteroaryl or aryl;

each (A′), if present, is independently chosen from aryl, arylalkoxy,arylalkyl, heterocyclyl, aryloxy, halo, alkoxy, haloalkyl, cycloalkyl,haloalkoxy, and cyano, wherein each (A′) is substituted with 0, 1, 2 or3 substituents independently chosen from halo, haloalkyl, aryl,arylalkoxy, alkyl, alkoxy, cyano, sulfonyl, sulfinyl, and carboxamide;

X is 0, 1, 2, or 3;

(B) is a cyclopropyl ring, wherein (A) and (Z) are covalently bonded todifferent carbon atoms of (B);

(Z) is —NH—; and

(L) is —(CH₂)_(m)CR₁R₂—, wherein m is 0, 1, 2, 3, 4, 5, or 6, andwherein R₁ and R₂ are each independently hydrogen or C₁-C₆ alkyl;

provided that, if (L) is —CH₂— or —CH(CH₃)—, then X is not 0.

Compounds of formula (D having a (trans) disposition on the substituentson the cyclopropyl ring are preferred.

Preferably, the compound of formula (D) is a compound from the listbelow:

-   2-((trans)-2-(4-(4-cyanobenzyloxy)phenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(4-(3-cyanobenzyloxy)phenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(4-(4-fluorobenzyloxy)phenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(4-(3-fluorobenzyloxy)phenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(4-(3-chlorobenzyloxy)phenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(4-(4-chlorobenzyloxy)phenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(4-(3-bromobenzyloxy)phenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(4-(3,5-difluorobenzyloxy)phenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(4-phenethoxyphenyl)cyclopropylamino)acetamide,-   2-((trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropylamino)acetamide,-   2-((trans)-2-(3′-chlorobiphenyl-4-yl)cyclopropylamino)acetamide,-   2-((trans)-2-(6-(4-chlorophenyl)pyridin-3-yl)cyclopropylamino)acetamide,-   (R)-2-((trans)-2-(4-(3-fluorobenzyloxy)phenyl)cyclopropylamino)propanamide,-   (S)-2-((trans)-2-(4-(4-fluorobenzyloxy)phenyl)cyclopropylamino)propanamide,-   (R)-2-((trans)-2-(4-(4-fluorobenzyloxy)phenyl)cyclopropylamino)propanamide,-   (S)-2-((trans)-2-(4-(4-fluorobenzyloxy)phenyl)cyclopropylamino)propanamide,-   (R)-2-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)propanamide,-   (S)-2-((trans)-2-(4-(benzyloxy)phenyl) cyclopropylamino)propanamide,-   2-(2-[1,1′;4′,1″]Terphenyl-4″-yl-cyclopropylamino)acetamide,-   5′-((trans)-2-(2-amino-2-oxoethylamino)cyclopropyl)-2′-(benzyloxy)biphenyl-3-carboxamide,-   5-((trans)-2-(4′-chlorobiphenyl-4-yl)cyclopropylamino)pentanamide,-   3-((trans)-2-(4-(3-bromobenzyloxy)phenyl)cyclopropylamino)propanamide,-   4-((trans)-2-phenylcyclopropylamino)butanamide,-   5-((trans)-2-phenylcyclopropylamino)pentanamide,-   5-((trans)-2-(4′-chlorobiphenyl-4-yl)cyclopropylamino)-2-methylpentanamide,-   4-((trans)-2-(4′-chlorobiphenyl-4-yl)cyclopropylamino)-2-methylbutanamide,-   3-((trans)-2-(4-(3-fluorobenzyloxy)phenyl)cyclopropylamino)-2,2-dimethylpropanamide,-   3-((trans)-2-(4′-chlorobiphenyl-4-yl)cyclopropylamino)propanamide,-   4-((trans)-2-(4′-chlorobiphenyl-4-yl)cyclopropylamino)butanamide,-   4-((trans)-2-(4-(3-bromobenzyloxy)phenyl)cyclopropylamino)butanamide,-   5-((trans)-2-(4-(3-bromobenzyloxy)phenyl)cyclopropylamino)pentanamide,-   5-((trans)-2-(6-(benzyloxy)biphenyl-3-yl)cyclopropylamino)pentanamide,    and-   4-((trans)-2-(6-(benzyloxy)biphenyl-3-yl)cyclopropylamino)butanamide,

and pharmaceutically acceptable salts thereof.

Compounds of formula (D) can be prepared by the methods disclosed inWO2011/042217, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (E) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

In formula (E), E is —N(R3)-, —O—, or —S—, or is —X₃═X⁴—;

X¹ and X² are independently C(R2) or N;

X³ and X⁴, when present, are independently C(R2) or N;

(G) is a cyclyl group;

each (R1) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl,-L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl;

each (R2) is independently chosen from —H, alkyl, alkenyl, alkynyl,cyclyl, -L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has1, 2, or 3 independently chosen optional substituents or two (R2) groupscan be taken together to form a heterocyclyl or aryl group having 1, 2,or 3 independently chosen optional substituents, wherein said optionalsubstituents are independently chosen from alkyl, alkanoyl, heteroalkyl,heterocyclyl, haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy,heterocyclylalkoxy, aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy,oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl,amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio,sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;

R3 is —H or a (C₁-C₆)alkyl group;

each L1 is independently alkylene or heteroalkylene; and

n is 0, 1, 2, 3, 4 or 5.

Compounds of formula (E) having a (trans) disposition on thesubstituents on the cyclopropyl ring are preferred.

Preferably, the compound of formula (E) is a compound from the listbelow:

-   (trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropanamine;-   (trans)-2-(terphenyl-4-yl)cyclopropanamine;-   4′-((trans)-2-aminocyclopropyl)biphenyl-4-ol;-   4′-((trans)-2-aminocyclopropyl)biphenyl-3-ol;-   (trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   (Trans)-2-(6-(3,5-dichlorophenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(4-chlorophenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(3-chlorophenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(4-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(4-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(3-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   4-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzonitrile;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzonitrile;-   (Trans)-2-(6-p-tolylpyridin-3-yl)cyclopropanamine;-   (Trans)-2-(6-m-tolylpyridin-3-yl)cyclopropanamine;-   4-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenol;-   4-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzamide;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzamide;-   2-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenol;-   (Trans)-2-(6-(3-methoxy-4-methylphenyl)pyridin-3-yl)cyclopropanamine;-   5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2-fluorophenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-fluorophenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-fluorophenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2-fluorophenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2,4-difluorophenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2,4,6-trifluorophenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-chlorophenol;-   (Trans)-2-(6-(2-fluoro-3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   (Trans)-2-(6-(5-chlorothiophen-2-yl)pyridin-3-yl)cyclopropanamine;-   (Trans)-2-(6-(5-methylthiophen-2-yl)pyridin-3-yl)cyclopropanamine;-   (Trans)-2-(6-(1H-indol-6-yl)pyridin-3-yl)cyclopropanamine;-   (Trans)-2-(6-(benzo[b]thiophen-5-yl)pyridin-3-yl)cyclopropanamine;-   3-(5-((trans)-2-aminocyclopropyl)-3-methylpyridin-2-yl)phenol;-   (trans)-2-(6-(3-chlorophenyl)-5-methylpyridin-3-yl)cyclopropanamine;-   (trans)-2-(5-methyl-6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(4-fluoro-3-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(3-fluoro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(2-fluoro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(2-fluoro-3-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(3-chloro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(2-chloro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(3-methoxy-5-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-methoxybenzonitrile;-   5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2-methylphenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-chlorophenol;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-(trifluoromethyl)phenol;-   (trans)-2-(6-(2-fluoro-5-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(2-chloro-5-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(3,5-bis(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)acetamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)methanesulfonamide;-   (trans)-2-(6-(benzo[b]thiophen-2-yl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(benzo[b]thiophen-3-yl)pyridin-3-yl)cyclopropanamine;-   5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)thiophene-2-carbonitrile;-   (trans)-2-(6-(4-methylthiophen-3-yl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(2-chloro-6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(2-(4-chlorophenyl)-6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   4-(3-((trans)-2-aminocyclopropyl)-6-(3-(trifluoromethyl)phenyl)pyridin-2-yl)phenol;-   4-(3-((trans)-2-aminocyclopropyl)-6-(3-(trifluoromethyl)phenyl)pyridin-2-yl)benzamide;-   (trans)-2-(2-methyl-6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-hydroxybenzonitrile;-   (trans)-2-(6-(3,4-difluoro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2,3-difluorophenol;-   (trans)-2-(6-(3-chloro-4-fluoro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;-   5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-3-chloro-2-fluorophenol;-   (trans)-2-(6-(1H-indazol-6-yl)pyridin-3-yl)cyclopropanamine;-   (trans)-2-(6-(9H-carbazol-2-yl)pyridin-3-yl)cyclopropanamine;-   6-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)indolin-2-one;-   6-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzofuran-2(3H)-one;-   4-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)pyridin-2(1H)-one;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)benzenesulfonamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)propane-2-sulfonamide;-   4′-((trans)-2-aminocyclopropyl)-4-fluorobiphenyl-3-ol;-   4′-((trans)-2-aminocyclopropyl)-5-chlorobiphenyl-3-ol;-   4′-((trans)-2-aminocyclopropyl)-5-chloro-4-fluorobiphenyl-3-ol;-   N-(4′-((trans)-2-aminocyclopropyl)biphenyl-3-yl)benzenesulfonamide;-   N-(4′-((trans)-2-aminocyclopropyl)biphenyl-3-yl)propane-2-sulfonamide;-   N-(4′-((trans)-2-aminocyclopropyl)biphenyl-3-yl)methanesulfonamide;-   N-(2-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)methanesulfonamide;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-methoxybenzonitrile;-   N-(4′-((trans)-2-aminocyclopropyl)biphenyl-2-yl)methanesulfonamide;-   4′-((trans)-2-aminocyclopropyl)-6-methoxybiphenyl-3-carbonitrile;-   N-(4′-((trans)-2-aminocyclopropyl)-6-methoxybiphenyl-3-yl)methanesulfonamide;-   4′-((trans)-2-aminocyclopropyl)-6-hydroxybiphenyl-3-carbonitrile;-   N-(4′-((trans)-2-aminocyclopropyl)-6-hydroxybiphenyl-3-yl)methanesulfonamide;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-hydroxybenzonitrile;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-hydroxyphenyl)methanesulfonamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-(trifluoromethyl)phenyl)methanesulfonamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-(trifluoromethyl)phenyl)methanesulfonamide;-   3-(6-((trans)-2-aminocyclopropyl)pyridin-3-yl)phenol;-   (Trans)-2-(5-(3-methoxyphenyl)pyridin-2-yl)cyclopropanamine;-   4-(6-((trans)-2-aminocyclopropyl)pyridin-3-yl)phenol;-   2-(6-((trans)-2-aminocyclopropyl)pyridin-3-yl)phenol;-   2-(5-((trans)-2-aminocyclopropyl)thiophen-2-yl)phenol;-   3-(5-((trans)-2-aminocyclopropyl)thiophen-2-yl)phenol;-   4-(5-((trans)-2-aminocyclopropyl)thiophen-2-yl)phenol;-   2-(5-((trans)-2-aminocyclopropyl)thiazol-2-yl)phenol;-   3-(5-((trans)-2-aminocyclopropyl)thiazol-2-yl)phenol;-   4-(5-((trans)-2-aminocyclopropyl)thiazol-2-yl)phenol;-   2-(2-((trans)-2-aminocyclopropyl)thiazol-5-yl)phenol;-   3-(2-((trans)-2-aminocyclopropyl)thiazol-5-yl)phenol;-   2-(2-((trans)-2-aminocyclopropyl)thiazol-5-yl)phenol;-   3-(2-((trans)-2-aminocyclopropyl)thiazol-5-yl)phenol;-   3-(5-((trans)-2-aminocyclopropyl)pyrimidin-2-yl)phenol;-   4-(5-((trans)-2-aminocyclopropyl)pyrimidin-2-yl)phenol;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-methoxyphenyl)methanesulfonamide;-   N-(4′-((trans)-2-aminocyclopropyl)-5-chloro-[1,1′-biphenyl]-3-yl)methanesulfonamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-chlorophenyl)methanesulfonamide;-   N-(4′-((trans)-2-aminocyclopropyl)-4-fluoro-[1,1′-biphenyl]-3-yl)methanesulfonamide;-   N-(5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2-fluorophenyl)methanesulfonamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)methanesulfonamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)-4-cyanobenzenesulfonamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)-3-cyanobenzenesulfonamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)-2-cyanobenzenesulfonamide;-   N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-(trifluoromethyl)phenyl)-4-cyanobenzenesulfonamide;-   N-(4′-((trans)-2-aminocyclopropyl)-[1,1′-biphenyl]-3-yl)-1,1,1-trifluoromethanesulfonamide;-   4′-((trans)-2-aminocyclopropyl)-6-hydroxy-[1,1′-biphenyl]-3-carbonitrile;-   4′-((trans)-2-aminocyclopropyl)-[1,1-biphenyl]-2-ol;-   4′-((trans)-2-aminocyclopropyl)-3′-methoxy-[1,1′-biphenyl]-3-ol;-   N-(3-(5-((trans)-2-aminocyclopropyl)thiazol-2-yl)phenyl)-2-cyanobenzenesulfonamide;

and pharmaceutically acceptable salts thereof.

Compounds of formula (E) can be prepared by the methods disclosed inWO2012/013727, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (F) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:(A′)_(x)-(A)-(B)—(Z)-(L)-(D)  (F)

In formula (F), (A) is heteroaryl or aryl;

each (A′), if present, is independently chosen from aryl, arylalkoxy,arylalkyl, heterocyclyl, aryloxy, halo, alkoxy, haloalkyl, cycloalkyl,haloalkoxy, and cyano, wherein each (A′) is substituted with 0, 1, 2, or3 substituents independently chosen from halo, haloalkyl, haloalkoxy,aryl, arylalkoxy, alkyl, alkoxy, amido, —CH₂C(═O)NH₂, heteroaryl, cyano,sulfonyl, and sulfinyl;

X is 0, 1, 2, or 3;

(B) is a cyclopropyl ring, wherein (A) and (Z) are covalently bonded todifferent carbon atoms of (B);

(Z) is —NH—;

(L) is chosen from a single bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, and—CH₂CH₂CH₂CH₂—; and

(D) is an aliphatic carbocyclic group or benzocycloalkyl, wherein saidaliphatic carbocyclic group or said benzocycloalkyl has 0, 1, 2, or 3substituents independently chosen from —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)(C₁-C₆ alkyl), alkyl, halo, amido, cyano, alkoxy, haloalkyl, andhaloalkoxy.

Preferably in formula (VI),

(A) is aryl or heteroaryl. Said aryl is preferably phenyl. Saidheteroaryl is preferably pyridinyl, pyrimidinyl, or thiophenyl; and/or

(A′), if present, is aryl or arylalkoxy. Said aryl is preferably phenyl.Said arylalkoxy is preferably benzyloxy, all of which can be optionallysubstituted as provided above; and/or

(L) is a single bond.

Compounds of formula (F) having a (trans) disposition on thesubstituents on the cyclopropyl ring are preferred.

Preferably, the compound of formula (F) is a compound from the listbelow:

-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)-6-methoxy-2,3-dihydro-1H-inden-1-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)-4,5-dimethoxy-2,3-dihydro-1H-inden-1-amine;-   N-((trans)-2-phenylcyclopropyl)-2,3-dihydro-1H-inden-1-amine;-   6-methoxy-N-((trans)-2-phenylcyclopropyl)-2,3-dihydro-1H-inden-1-amine;-   6-chloro-N-((trans)-2-phenylcyclopropyl)-2,3-dihydro-1H-inden-1-amine;-   N-((trans)-2-phenylcyclopropyl)-6-(trifluoromethyl)-2,3-dihydro-1H-inden-1-amine;-   7-methoxy-N-((trans)-2-phenylcyclopropyl)-1,2,3,4-tetrahydronaphthalen-1-amine;-   N-((trans)-2-(3′-chlorobiphenyl-4-yl)cyclopropyl)-6-methoxy-2,3-dihydro-1H-inden-1-amine;-   N-((trans)-2-(4′-chlorobiphenyl-4-yl)cyclopropyl)-6-methoxy-2,3-dihydro-1H-inden-1-amine;-   6-methoxy-N-((trans)-2-(3′-methoxybiphenyl-4-yl)cyclopropyl)-2,3-dihydro-1H-inden-1-amine;-   N-trans-(2-cyclohexylethyl)-2-phenylcyclopropanamine;-   (Trans)-N-(3-cyclohexylpropyl)-2-phenylcyclopropanamine;-   (Trans)-N-(2-cycloheptylethyl)-2-phenylcyclopropanamine;-   (Trans)-2-(4-(3-bromobenzyloxy)phenyl)-N-(2-cyclohexylethyl)    cyclopropanamine;-   N-((trans)-2-(4-(3-bromobenzyloxy)phenyl)cyclopropyl)-6-methoxy-2,3-dihydro-1H-inden-1-amine;-   (Trans)-2-(3′-chlorobiphenyl-4-yl)-N-(2-cyclohexylethyl)cyclopropanamine;-   (Trans)-2-(4′-chlorobiphenyl-4-yl)-N-(2-cyclohexylethyl)cyclopropanamine;-   (Trans)-N-(2-cyclohexylethyl)-2-(3′-methoxybiphenyl-4-yl)cyclopropanamine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1-amine;    and-   1-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)cyclopropanecarboxamide;

and pharmaceutically acceptable salts thereof.

Compounds of formula (F) can be prepared by the methods disclosed inWO2011/131697, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (G) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

In formula (G), E is —X³═X⁴—, —N(R3)-, —S—, or —O—,

X¹ and X² are each independently C(R2) or N;

X³ and X⁴, when present, are each independently C(R2) or N;

L1 is —NH— or —NH—CH₂—;

G is a cyclyl group;

each R1 is independently chosen from alkyl, alkenyl, alkynyl, cyclyl,-L2-cyclyl, -L2-amino, -L2-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl;

each R2 is independently chosen from —H, alkyl, alkenyl, alkynyl,cyclyl, -L2-cyclyl, -L2-amino, -L2-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl, wherein each R2 group has 1,2, or 3 independently chosen optional substituents, and further whereintwo R2 groups bound to adjacent carbon atoms can be taken together toform a heterocyclyl or aryl group having 1, 2, or 3 independently chosenoptional substituents; wherein said optional substituents are eachindependently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl,haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy, heterocyclylalkoxy,aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy,carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino,aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio,sulfinyl, sulfonyl, sulfonamide, urea or carbamate;

R3 is —H or an (C1-C6)alkyl group;

each L2 is independently chosen from alkylene or heteroalkylene; and

n is 0, 1, 2, 3, 4 or 5.

Compounds of formula (G) having a (trans) disposition on thesubstituents on the cyclopropyl ring are preferred.

Preferably, the compound of formula (G) is a compound from the listbelow:

-   5-((trans)-2-aminocyclopropyl)-N-(3-chlorophenyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(4-chlorophenyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(4-(trifluoromethyl)phenyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(3-methoxyphenyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(4-methoxyphenyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-p-tolylpyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-m-tolylpyridin-2-amine;-   4-(5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)benzonitrile;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)benzonitrile;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)benzamide;-   4-(5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)benzamide;-   5-((trans)-2-aminocyclopropyl)-N-(3-chlorobenzyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(4-chlorobenzyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(3-(trifluoromethyl)benzyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(4-(trifluoromethyl)benzyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(3-methylbenzyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(4-methylbenzyl)pyridin-2-amine;-   3-((5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)methyl)benzonitrile;-   4-((5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)methyl)benzonitrile;-   5-((trans)-2-aminocyclopropyl)-N-(3-methoxybenzyl)pyridin-2-amine;-   5-((trans)-2-aminocyclopropyl)-N-(4-methoxybenzyl)pyridin-2-amine;-   4-(5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)phenol;-   3-((5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)methyl)benzamide;-   4-((5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)methyl)benzamide;-   4-((5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)methyl)phenol;-   5-((trans)-2-aminocyclopropyl)-N-(3-ethynylphenyl)pyridin-2-amine;-   N-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-1H-indol-7-amine;-   N-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-1H-indazol-7-amine;-   3-(5-((trans)-2-aminocyclopropyl)pyridin-2-ylamino)phenol;-   4-((trans)-2-aminocyclopropyl)-N-(4-methylbenzyl)aniline;-   4-((trans)-2-aminocyclopropyl)-N-(4-(trifluoromethyl)benzyl)aniline;-   4-((trans)-2-aminocyclopropyl)-N-(3-chlorobenzyl)aniline;-   3-(((4-((trans)-2-aminocyclopropyl)phenyl)amino)methyl)benzonitrile;-   4-((trans)-2-aminocyclopropyl)-N-(p-tolyl)aniline;-   4-((trans)-2-aminocyclopropyl)-N-(4-chlorophenyl)aniline;-   3-((4-((trans)-2-aminocyclopropyl)phenyl)amino)benzonitrile;-   N-(4-((trans)-2-aminocyclopropyl)phenyl)-3-methoxyaniline;-   3-((4-((trans)-2-aminocyclopropyl)phenyl)amino)benzamide;

and pharmaceutically acceptable salts thereof.

Compounds of formula (G) can be prepared by the methods disclosed inWO2012/045883, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (H) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

In formula (H), (A) is a cyclyl group having n substituents (R3);

(B) is a cyclyl group or an -(L1)-cyclyl group, wherein said cyclylgroup or the cyclyl moiety comprised in said -(L1)-cyclyl group has nsubstituents (R2);

(L1) is —O—, —NH—, —N(alkyl)-, alkylene or heteroalkylene;

(D) is a heteroaryl group or an -(L2)-heteroaryl group, wherein saidheteroaryl group or the heteroaryl moiety comprised in said-(L2)-heteroaryl group has one substituent (R1), and further whereinsaid heteroaryl group is covalently bonded to the remainder of themolecule through a ring carbon atom or the heteroaryl moiety comprisedin said -(L2)-heteroaryl group is covalently bonded to the (L2) moietythrough a ring carbon atom;

(L2) is —O—, —NH—, —N(alkyl)-, alkylene or heteroalkylene;

(R1) is a hydrogen bonding group, including but not limited to —OH,—NH₂, amido, —S(O)₂NH₂, —C(═O)NH₂, —CH₂—C(═O)NH₂, —NH—C(═O)CH₃, —NHCH₃,—N(CH₃)₂ or —CH₂—NH₂;

each (R2) is independently selected from alkyl, alkenyl, alkynyl,cyclyl, amino, amido, C-amido, alkylamino, hydroxyl, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, alkoxy,acyl, carboxyl, carbamate or urea;

each (R3) is independently selected from alkyl, alkenyl, alkynyl,cyclyl, amino, amido, C-amido, alkylamino, hydroxyl, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, alkoxy,acyl, carboxyl, carbamate, or urea; and

n is independently 0, 1, 2, 3 or 4.

Preferably in formula (H),

(A) is aryl or heteroaryl. Said aryl is preferably phenyl. Saidheteroaryl is preferably pyridinyl, and/or;

(B) is —O—CH₂-phenyl or phenyl, each of which can be optionallysubstituted with n substituents R2, and/or;

(D) is a monocyclic heteroaryl, preferably thiazolyl, oxadiazolyl orpyrimidinyl, and more preferably oxadiazolyl; and/or;

(R1) is —NH₂ or —NHCH₃ and more preferably —NH₂.

Compounds of formula (H) having a (trans) disposition on thesubstituents on the cyclopropyl ring are preferred.

Preferably, the compound of formula (H) is a compound from the listbelow:

-   5-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)pyrimidin-2-amine;-   5-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)thiazol-2-amine;-   5-(((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropylamino)methyl)pyrimidin-2-amine;-   5-(((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropylamino)methyl)thiazol-2-amine;-   3-(5-((trans)-2-((2-aminopyrimidin-5-yl)methylamino)cyclopropyl)pyridin-2-yl)phenol;-   3-(5-((trans)-2-((2-aminothiazol-5-yl)methylamino)cyclopropyl)pyridin-2-yl)phenol;-   4′-((trans)-2-((2-aminopyrimidin-5-yl)methylamino)cyclopropyl)biphenyl-3-ol;-   4′-((trans)-2-((2-aminothiazol-5-yl)methylamino)cyclopropyl)biphenyl-3-ol;-   5-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)-1,2,4-oxadiazol-3-amine;-   5-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((4-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((3-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((3,5-difluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((4-chlorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((3-chlorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((2-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-N-methyl-1,3,4-oxadiazol-2-amine;-   N-(5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-yl)acetamide;-   4′-((trans)-2-(((5-amino-1,3,4-oxadiazol-2-yl)methyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-ol;-   5-((((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-thiadiazol-2-amine;-   2-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)thiazol-5-amine;-   4-((((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)amino)methyl)thiazol-2-amine;-   2-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)oxazol-5-amine;-   3-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)isoxazol-5-amine;-   5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-N,N-dimethyl-1,3,4-oxadiazol-3-amine;-   3-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,2,4-oxadiazol-5-amine;-   5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,2,4-thiadiazol-3-amine;-   5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)pyridin-2-amine;-   6-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)pyridazin-3-amine;-   5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)pyrazin-2-amine;-   2-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)pyrimidin-5-amine;-   6-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,2,4-triazin-3-amine;-   3-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,2,4-triazin-6-amine;-   4′-((trans)-2-((2-aminothiazol-5-yl)methylamino)cyclopropyl)biphenyl-3-ol;-   5-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)-1,2,4-oxadiazol-3-amine;-   5-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((4-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((3-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   (−)5-(((trans)-2-(4-((3-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-N-methyl-1,3,4-oxadiazol-2-amine;-   (−)N-(5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-yl)acetamide;-   (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)pyrimidin-2-amine;-   (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-thiadiazol-2-amine;-   (−)5-((((trans)-2-(4-((2-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   4′-((trans)-2-((2-aminothiazol-5-yl)methylamino)cyclopropyl)biphenyl-3-ol;-   5-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)-1,2,4-oxadiazol-3-amine;-   5-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((4-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   5-((((trans)-2-(4-((3-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   (−)5-((((trans)-2-(4-((3-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-N-methyl-1,3,4-oxadiazol-2-amine;-   (−)N-(5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-yl)acetamide;-   (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)pyrimidin-2-amine;-   (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-thiadiazol-2-amine;-   (−)5-((((trans)-2-(4-((2-fluorobenzyl)oxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;

and pharmaceutically acceptable salts thereof.

Still more preferably, the compound of formula (H) is(−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine,or a pharmaceutically acceptable salt thereof.

Compounds of formula (H) can be prepared by the methods disclosed inWO2012/013728, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (J) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

wherein:

A is aryl or heteroaryl, wherein said aryl or said heteroaryl isoptionally substituted with one or more R¹;

B is hydrogen, R¹ or -L-E;

E is aryl or heteroaryl, wherein said aryl or said heteroaryl isoptionally substituted with one or more R²;

L is a bond, —O—, —NH—, —N(C₁₋₄ alkyl)-, C₁₋₄ alkylene or heteroC₁₋₄alkylene;

D is a cycloalkyl group having from 4 to 7 C atoms, wherein saidcycloalkyl group has one or two substituents R³ and is furtheroptionally substituted with one or more R⁴, and wherein the cycloalkylgroup optionally:

-   -   (a) is fused to a phenyl or a 5- or 6-membered aromatic        heterocyclic ring containing from 1 to 3 heteroatoms        independently selected from N, O and S, wherein said fused        phenyl or said fused aromatic heterocyclic ring is optionally        substituted with one or more R⁵; or    -   (b) is bonded to a linker group —(C(R^(a))₂)_(p)— linking        together any two non-adjacent ring carbon atoms of the        cycloalkyl group, wherein p is 1 or 2 and each R^(a)        independently is hydrogen or C₁₋₄ alkyl; or    -   (c) is linked to a second ring that is either a 3- to 7-membered        saturated carbocyclic ring or a 3- to 7-membered saturated        heterocyclic ring containing from 1 to 3 heteroatoms        independently selected from N, O and S, wherein said second ring        is linked together with the cycloalkyl group via a single carbon        atom common to both rings, and wherein said second ring is        optionally substituted with one or more R⁶;

each R¹ is independently selected from C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, cyclyl, amino, amido, hydroxyl, nitro, halo, haloC₁₋₈ alkyl,haloC₁₋₈ alkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, C₁₋₈ alkoxy,acyl, carboxyl, O-carboxy, C-carboxy, carbamate and urea;

each R² is independently selected from C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, cyclyl, amino, amido, hydroxyl, nitro, halo, haloC₁₋₈ alkyl,haloC₁₋₈ alkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, C₁₋₈ alkoxy,acyl, carboxyl, O-carboxy, C-carboxy, carbamate and urea;

each R³ is independently selected from —NR⁷R⁸, —NHOH, —NR⁹COR¹⁰,—NR⁹SO₂R¹⁰, —NR⁹COOR¹⁰, —NR⁹CONR⁷R⁸, —NR⁹SO₂NR⁷R⁸, —OH, —CONR⁷R⁸, oxo,—C₁₋₄alkylene-NR⁷R⁸, —C₁₋₄ alkylene-NHOH, —C₁₋₄ alkyene-NR⁹COR¹⁰, —C₁₋₄alkylene-NR⁹SO₂R¹⁰, —C₁₋₄ alkylene-NR⁹COOR¹⁰, —C₁₋₄ alkylene-NR⁹CONR⁷R⁸,—C₁₋₄ alkylene-NR⁹SO₂NR⁷R⁸, —C₁₋₄alkylene-OH and —C₁₋₄alkylene-CONR⁷R⁸;

each R⁴ and each R⁶ is independently selected from C₁₋₈ alkyl, halo,haloC₁₋₈ alkyl, haloC₁₋₈ alkoxy and C₁₋₈ alkoxy;

each R⁵ is independently selected from C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, cyclyl, amino, amido, hydroxyl, nitro, halo, haloC₁₋₈ alkyl,haloC₁₋₈ alkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, C₁₋₈ alkoxy,acyl, carboxyl, O-carboxy, C-carboxy, carbamate and urea;

each R⁷ and each R⁸ is independently selected from hydrogen, C₁₋₈ alkyl,R¹²R¹³N—C₁₋₈ alkyl and hydroxyC₁₋₈ alkyl, or R⁷ and R⁸ are linkedtogether to form, along with the N atom to which they are bound, asaturated 3- to 7-membered heterocyclic ring which optionally containsone further heteroatom selected from N, O and S, wherein one or more Catoms in said heterocyclic ring are optionally oxidized to form COgroups, wherein one or more S atoms in said heterocyclic ring, ifpresent, are optionally oxidized to form independently SO groups or SO₂groups, and wherein said heterocyclic ring is optionally substitutedwith one or more R¹¹;

each R⁹ is independently selected from hydrogen and C₁₋₄ alkyl;

each R¹⁰ is independently selected from C₁₋₈ alkyl, haloC₁₋₈ alkyl,cyclyl and cyclylC₁₋₈ alkyl, wherein said cyclyl or the cyclyl moietycomprised in said cyclylC₁₋₈ alkyl is optionally substituted with one ormore R¹⁴;

each R¹¹ is independently selected from C₁₋₈ alkyl, halo, C₁₋₈ alkoxy,hydroxyl and —NR¹²R¹³;

each R¹² and each R¹³ is independently selected from hydrogen and C₁₋₈alkyl;

each R¹⁴ is independently selected from C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, amino, amido, hydroxyl, nitro, halo, haloC₁₋₈ alkyl, haloC₁₋₈alkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, C₁₋₈ alkoxy, acyl,carboxyl, O-carboxy, C-carboxy, carbamate and urea; and

each R^(w), R^(x), R^(y) and R^(z) is independently selected fromhydrogen, halo and C₁₋₄ alkyl.

Preferably in formula (J),

(A) is phenyl, thiazolyl or pyridyl, preferably phenyl, which rings canbe optionally substituted with one or more R1, and/or

(B) is H, and/or

(R1) is Cm alkyl, amino, amido, hydroxyl, halo, haloC₁₋₈ alkyl,haloC₁₋₈alkoxy, cyano, sulfonamide, C₁₋₈ alkoxy, acyl, carboxyl,carbamate, and urea, and more preferably halo, C₁₋₄ alkyl, haloC₁₋₄alkyl, C₁₋₄ alkoxy and C₃₋₆ cycloalkyl; and/or

(D) is selected from D1, D2, D3 and D4:

and more preferably D3; and/or

(R3) is selected from —NR⁷R⁸, —NHOH, —NR⁹COR¹⁰, —NR⁹SO₂R¹⁰, —NR⁹COOR¹⁰,—NR⁹CONR⁷R⁸, —NR⁹SO₂NR⁷R⁸, —OH, —CONR⁷R⁸, oxo, —C₁₋₄ alkylene-NR⁷R⁸,—C₁₋₄ alkylene-OH and —C₁₋₄ alkylene-CONR⁷R⁸, more preferably from—NR⁷R⁸, —OH, —C₁₋₄ alkylene-NR⁷R⁸, and —C₁₋₄ alkylene-OH, still morepreferably —NR⁷R⁸ (such as —NH2); and/or

each R^(w), R^(x), R^(y) and R^(z) is hydrogen.

Compounds of formula (J) having a (trans) disposition on thesubstituents on the cyclopropyl ring are preferred. Preferably, thecompound of formula (J) is a compound from the list below:

-   N1-((trans)-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1S,2R)-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1S,2R)-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1R,2S)-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1R,2S)-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(thiazol-5-yl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(pyridin-3-yl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   4-(((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropyl)amino)cyclohexanol;-   4-(((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropyl)amino)cyclohexanecarboxamide;-   N-(4-(((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropyl)amino)cyclohexyl)acetamide;-   N-(4-(((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropyl)amino)cyclohexyl)methanesulfonamide;-   (R)-1-(4-(((trans)-2-phenylcyclopropyl)amino)cyclohexyl)pyrrolidin-3-amine;-   N1-((trans)-2-(4′-chloro-[1,1′-biphenyl]-4-yl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(3′-chloro-[1,1′-biphenyl]-4-yl)cyclopropyl)cyclohexane-1,4-diamine;-   4′-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-ol;-   N-(4′-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)methanesulfonamide;-   N1-((trans)-2-(4-((2-fluorobenzyl)oxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-((3-fluorobenzyl)oxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-((4-fluorobenzyl)oxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-methyl-N4-((trans)-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   N1-methyl-N4-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)-N4-methylcyclohexane-1,4-diamine;-   N1-((trans)-2-phenylcyclopropyl)cyclobutane-1,3-diamine;-   N1-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)cyclobutane-1,3-diamine;-   N1-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)cyclobutane-1,3-diamine;-   N1-((trans)-2-phenylcyclopropyl)-2,3-dihydro-1H-indene-1,3-diamine;-   N1-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)-2,3-dihydro-1H-indene-1,3-diamine;-   N1-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)-2,3-dihydro-1H-indene-1,3-diamine;-   N1-((trans)-2-fluoro-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   N1-((1S,2S)-2-fluoro-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   N1-((1R,2R)-2-fluoro-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   1-methyl-N4-((trans)-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   4-(aminomethyl)-N-((trans)-2-phenylcyclopropyl)cyclohexanamine;-   N1-((trans)-2-phenylcyclopropyl)cyclohexane-1,3-diamine;-   N1-((cis)-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   Tert-butyl    (4-(((trans)-2-phenylcyclopropyl)amino)cyclohexyl)carbamate;-   1-ethyl-3-(4-(((trans)-2-phenylcyclopropyl)amino)cyclohexyl)urea;-   4-morpholino-N-((trans)-2-phenylcyclopropyl)cyclohexanamine;-   N1-((trans)-2-(4-bromophenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-(2-(o-tolyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-(2-(4-(trifluoromethyl)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-(2-(4-methoxyphenyl)cyclopropyl)cyclohexane-1,4-diamine;-   4-(2-((4-aminocyclohexyl)amino)cyclopropyl)phenol;-   N1-(2-(2-fluorophenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-(2-(3,4-difluorophenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-(2-(naphthalen-2-yl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-(2-methyl-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   (R)-1-(4-(((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)    amino)cyclohexyl)pyrrolidin-3-amine;-   (Cis)-N1-((1S,2R)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)cyclohexane-1,4-diamine;-   (Trans)-N1-((1S,2R)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclo-propyl)cyclohexane-1,4-diamine;-   (Cis)-N1-((1R,2S)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclo-propyl)cyclohexane-1,4-diamine;-   (Trans)-N1-((1R,2S)-2-(3′-(trifluoromethyl)-[1,1-biphenyl]-4-yl)cyclo-propyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-cyclopropylphenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-(pyridin-3-yl)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-(1H-indazol-6-yl)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-(1H-pyrazol-5-yl)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   3-(5-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)thiophen-2-yl)phenol;-   3-(5-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)thiazol-2-yl)phenol;-   3-(5-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)pyridin-2-yl)-5-methoxybenzonitrile;-   5-(5-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)pyridin-2-yl)-2-methylphenol;-   N-(4′-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)-6-methoxy-[1,1′-biphenyl]-3-yl)methanesulfonamide;-   N-(3-(5-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)thiazol-2-yl)phenyl)-2-cyanobenzenesulfonamide;-   N-(4′-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)-2-cyanobenzenesulfonamide;-   6-amino-N-(4′-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)pyridine-3-sulfonamide;-   N-(4′-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)piperazine-1-sulfonamide;-   N1-((cis)-2-fluoro-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-((3-(piperazin-1-yl)benzyl)oxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-(pyridin-3-ylmethoxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(6-((3-methylbenzyl)amino)pyridin-3-yl)cyclopropyl)cyclohexane-1,4-diamine;-   3-((5-((trans)-2-((4-aminocyclohexyl)amino)cyclopropyl)pyridin-2-yl)    amino)benzonitrile;-   N1-((trans)-2-(naphthalen-2-yl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(o-tolyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-(trifluoromethyl)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(4-methoxyphenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(2-fluorophenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-(3,4-difluorophenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N1-((trans)-2-methyl-2-phenylcyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1S,2R)-2-(pyridin-3-yl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1R,2S)-2-(pyridin-3-yl)cyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1R,2S)-2-(pyridin-3-yl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1S,2R)-2-(pyridin-3-yl)cyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1S,2R)-2-phenylcyclopropyl)cyclobutane-1,3-diamine;-   (trans)-N1-((1R,2S)-2-phenylcyclopropyl)cyclobutane-1,3-diamine;-   (cis)-N1-((1R,2S)-2-phenylcyclopropyl)cyclobutane-1,3-diamine;-   (trans)-N1-((1S,2R)-2-phenylcyclopropyl)cyclobutane-1,3-diamine;-   (cis)-N1-((1S,2R)-2-(3,4-difluorophenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1S,2R)-2-(3,4-difluorophenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1S,2R)-2-(naphthalen-2-yl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1R,2S)-2-(naphthalen-2-yl)cyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1R,2S)-2-(naphthalen-2-yl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1S,2R)-2-(naphthalen-2-yl)cyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1S,2R)-2-(4-(1H-pyrazol-5-yl)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1R,2S)-2-(4-(1H-pyrazol-5-yl)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1R,2S)-2-(4-(1H-pyrazol-5-yl)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1S,2R)-2-(4-(1H-pyrazol-5-yl)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   N-(4′-((1R,2S)-2-(((cis)-4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)piperazine-1-sulfonamide;-   N-(4′-((1S,2R)-2-(((trans)-4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)piperazine-1-sulfonamide;-   N-(4′-((1S,2R)-2-(((cis)-4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3yl)piperazine-1-sulfonamide;-   N-(4′-((1R,2S)-2-(((trans)-4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3yl)piperazine-1-sulfonamide;-   (cis)-N1-((1S,2R)-2-(4-((2-fluorobenzyl)oxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1R,2S)-2-(4-((2-fluorobenzyl)oxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (cis)-N1-((1R,2S)-2-(4-((2-fluorobenzyl)oxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;-   (trans)-N1-((1S,2R)-2-(4-((2-fluorobenzyl)oxy)phenyl)cyclopropyl)cyclohexane-1,4-diamine;

and pharmaceutically acceptable salts thereof.

Still more preferably, the compound of formula (J) is(trans)-N1-((1R,2S)-2-phenylcyclopropyl)cyclohexane-1,4-diamine; or apharmaceutically acceptable salt thereof.

Compounds of formula (J) can be prepared by the methods disclosed inWO2013/057322, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (K) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

wherein:

A is aryl or heteroaryl, wherein said aryl or heteroaryl is optionallysubstituted with one or more R¹;

B is H, R¹ or -L¹-E;

E is aryl or heteroaryl; wherein said aryl or said heteroaryl isoptionally substituted with one or more R²;

L¹ is a bond, —O—, —NH—, —N(C₁₋₄ alkyl)-, C₁₋₄ alkylene orheteroC₁₋₄alkylene;

L² is a bond and D is a cyclic group selected from:

-   -   (i) a 3- to 7-membered monocyclic saturated heterocyclic ring        containing 1 or 2 heteroatoms independently selected from N, O        and S, and    -   (ii) a 7- to 15-membered polycyclic ring system which comprises        at least one saturated heterocyclic ring, wherein the polycyclic        ring system contains from 1 to 4 heteroatoms independently        selected from N, O and S,

wherein the cyclic group (i) or (ii) is linked to the remainder of thecompound of Formula I through a ring C atom,

wherein one or more ring C atoms in the cyclic group (i) or (ii) areoptionally oxidized to form CO groups, wherein one or more S atoms inthe cyclic group (i) or (ii), if present, are optionally oxidized toform independently SO groups or SO₂ groups, and

wherein the cyclic group (i) or (ii) is optionally substituted with oneor more R³;

or L² is C₁₋₄ alkylene and D is a cyclic group selected from:

-   -   (iii) a 3- to 7-membered monocyclic saturated heterocyclic ring        containing 1 or 2 heteroatoms independently selected from N, O        and S, and    -   (iv) a 7- to 15-membered polycyclic saturated ring system which        comprises at least one heterocyclic ring, wherein the polycyclic        saturated ring system contains from 1 to 4 heteroatoms        independently selected from N, O and S,

wherein the cyclic group (iii) or (iv) is linked to the remainder of thecompound of Formula I through a ring C atom,

wherein one or more ring C atoms in the cyclic group (iii) or (iv) areoptionally oxidized to form CO groups,

wherein one or more S atoms in the cyclic group (iii) or (iv), ifpresent, are optionally oxidized to form independently SO groups or SO₂groups, and

wherein the cyclic group (iii) or (iv) is optionally substituted withone or more R³;

each R¹ is independently selected from C₁₋₈ alkyl, C₂₋₈ alkenyl,alkynyl, cyclyl, amino, amido, hydroxyl, nitro, halo, haloC₁₋₈ alkyl,haloC₁₋₈ alkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, C₁₋₈ alkoxy,acyl, carboxyl, O-carboxy, C-carboxy, carbamate and urea;

each R² is independently selected from C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, cyclyl, amino, amido, hydroxyl, nitro, halo, haloC₁₋₈ alkyl,haloC₁₋₈ alkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, C₁₋₈ alkoxy,acyl, carboxyl, O-carboxy, C-carboxy, carbamate and urea;

each R³ is independently selected from C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, cyclyl, amino, amido, hydroxyl, nitro, halo, haloC₁₋₈ alkyl,haloC₁₋₈ alkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, C₁₋₈ alkoxy,acyl, carboxyl, O-carboxy, C-carboxy, carbamate and urea; and

each R^(w), R^(x), R^(y) and R^(z) is independently selected fromhydrogen, halo and C₁₋₄ alkyl.

Preferably in formula (K),

(A) is phenyl, thiazolyl or pyridyl, preferably phenyl, which rings canbe optionally substituted with one or more R1, and/or

(B) is H, and/or

(R¹) is C₁₋₈ alkyl, amino, amido, hydroxyl, halo, haloC₁₋₈ alkyl,haloC₁₋₈alkoxy, cyano, sulfonamide, C₁₋₈ alkoxy, acyl, carboxyl,carbamate, and urea and more preferably halo, C₁₋₄ alkyl, haloC₁₋₄alkyl, C₁₋₄ alkoxy and C₃₋₈ cycloalkyl; and/or

L2 is a bond and (D) is a 3- to 7-membered monocyclic saturatedheterocyclic ring containing 1 heteroatom selected from N, O and Swherein D is linked to the remainder of the compound of formula (X)through a C, more preferably a 3- to 7-membered monocyclic saturatedheterocyclic ring containing 1 N atom wherein D is linked to theremainder of the compound of formula (X) through a C, and even morepreferably D is 4-piperidinyl, or L2 is a bond and (D) is a ring systemselected from (a), (b), (c) and (d)

wherein any D is optionally substituted with one or more R3; and/or

each R^(w), R^(x), R^(y) and R^(z) is hydrogen.

Compounds of formula (K) having a (trans) disposition on thesubstituents on the cyclopropyl ring are preferred.

Preferably, the compound of formula (K) is a compound from the listbelow:

-   N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   N-((1S,2R)-2-phenylcyclopropyl)piperidin-4-amine;-   N-((1R,2S)-2-phenylcyclopropyl)piperidin-4-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropyl)tetrahydro-2H-pyran-4-amine;-   N-((trans)-2-(pyridin-3-yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(thiazol-5-yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-phenylcyclopropyl)piperidin-3-amine;-   N-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)piperidin-3-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)piperidin-3-amine;-   N-((trans)-2-phenylcyclopropyl)pyrrolidin-3-amine;-   N-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)pyrrolidin-3-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)pyrrolidin-3-amine;-   N-((trans)-2-phenylcyclopropyl)azetidin-3-amine;-   N-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)azetidin-3-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)azetidin-3-amine;-   N-((trans)-2-phenylcyclopropyl)azepan-3-amine;-   N-((trans)-2-phenylcyclopropyl)-8-azabicyclo[3.2.1]octan-3-amine;-   N-((trans)-2-phenylcyclopropyl)-3-azabicyclo[3.2.1]octan-8-amine;-   N-((trans)-2-phenylcyclopropyl)decahydroquinolin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-1,2,3,4-tetrahydroquinolin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-3-azaspiro[5.5]undecan-9-amine;-   N-((trans)-2-phenylcyclopropyl)-2-azaspiro[4.5]decan-8-amine;-   N-((trans)-2-phenylcyclopropyl)-2,3-dihydrospiro[indene-1,4′-piperidin]-3-amine;-   N-((1S,2R)-2-(4-(benzyloxy)phenyl)cyclopropyl)piperidin-4-amine;-   N-((1R,2S)-2-(4-(benzyloxy)phenyl)cyclopropyl)piperidin-4-amine;-   N-((1S,2R)-2-(pyridin-3-yl)cyclopropyl)piperidin-4-amine;-   N-((1R,2S)-2-(pyridin-3-yl)cyclopropyl)piperidin-4-amine;-   N-((1S,2S)-2-(thiazol-5-yl)cyclopropyl)piperidin-4-amine;-   N-((1R,2R)-2-(thiazol-5-yl)cyclopropyl)piperidin-4-amine;-   N-((1S,2R)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)piperidin-4-amine;-   N-((1R,2S)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-7-azaspiro[3.5]nonan-2-amine;-   N-(2-(o-tolyl)cyclopropyl)piperidin-4-amine;-   N-(2-(2-fluorophenyl)cyclopropyl)piperidin-4-amine;-   N-(2-(3,4-difluorophenyl)cyclopropyl)piperidin-4-amine;-   N-(2-(4-methoxyphenyl)cyclopropyl)piperidin-4-amine;-   N-(2-(naphthalen-2-yl)cyclopropyl)piperidin-4-amine;-   N-(2-methyl-2-phenylcyclopropyl)piperidin-4-amine;-   N-(6-methoxy-4′-((trans)-2-(piperidin-4-ylamino)cyclopropyl)-[1,1′-biphenyl]-3-yl)methanesulfonamide;-   N-(4′-((trans)-2-(piperidin-4-ylamino)cyclopropyl)-[1,1′-biphenyl]-3-yl)propane-2-sulfonamide;-   1-(methylsulfonyl)-N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   1-(4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)piperidin-1-yl)ethanone;-   4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)piperidine-1-carboxamide;-   N-((trans)-2-(4-bromophenyl)cyclopropyl)tetrahydro-2H-pyran-4-amine;-   2,2,6,6-tetramethyl-N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   1-methyl-N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   1-isopropyl-N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-1-(2,2,2-trifluoroethyl)piperidin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-1-(pyridin-4-yl)piperidin-4-amine;-   4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)tetrahydro-2H-thiopyran    1,1-dioxide;-   N-((trans)-2-fluoro-2-phenylcyclopropyl)piperidin-4-amine;-   N-((1S,2S)-2-fluoro-2-phenylcyclopropyl)piperidin-4-amine;-   N-((1R,2R)-2-fluoro-2-phenylcyclopropyl)piperidin-4-amine;-   N-((trans)-2-(naphthalen-2-yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-methyl-2-phenylcyclopropyl)piperidin-4-amine;-   N-((trans)-2-(o-tolyl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(2-fluorophenyl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(3,4-difluorophenyl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(4-methoxyphenyl)cyclopropyl)piperidin-4-amine;-   (Trans)-2-phenyl-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (Trans)-2-phenyl-N-(2-(piperidin-4-yl)ethyl)cyclopropanamine;-   (Trans)-2-phenyl-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)cyclopropanamine;-   (Trans)-2-(4′-chloro-[1,1′-biphenyl]-4-yl)-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)cyclopropanamine;-   (Trans)-N-(piperidin-4-ylmethyl)-2-(pyridin-3-yl)cyclopropanamine;-   (Trans)-N-(piperidin-4-ylmethyl)-2-(thiazol-5-yl)cyclopropanamine;-   (Trans)-N-(piperidin-4-ylmethyl)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropanamine;-   (Trans)-2-(4-(benzyloxy)phenyl)-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (Trans)-N-(2-(piperidin-4-yl)ethyl)-2-(pyridin-3-yl)cyclopropanamine;-   (Trans)-N-(2-(piperidin-4-yl)ethyl)-2-(thiazol-5-yl)cyclopropanamine;-   (Trans)-N-(2-(piperidin-4-yl)ethyl)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropanamine;-   (Trans)-2-(4-(benzyloxy)phenyl)-N-(2-(piperidin-4-yl)ethyl)cyclopropanamine;-   (1S,2R)-2-phenyl-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (1R,2S)-2-phenyl-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (1S,2R)-2-phenyl-N-(2-(piperidin-4-yl)ethyl)cyclopropanamine;-   (1R,2S)-2-phenyl-N-(2-(piperidin-4-yl)ethyl)cyclopropanamine;-   (1S,2R)—N-(piperidin-4-ylmethyl)-2-(pyridin-3-yl)cyclopropanamine;-   (1R,2S)—N-(piperidin-4-ylmethyl)-2-(pyridin-3-yl)cyclopropanamine;-   (1S,2S)—N-(piperidin-4-ylmethyl)-2-(thiazol-5-yl)cyclopropanamine;-   (1R,2R)—N-(piperidin-4-ylmethyl)-2-(thiazol-5-yl)cyclopropanamine;-   (1S,2R)—N-(piperidin-4-ylmethyl)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4yl)cyclopropanamine;-   (1R,2S)—N-(piperidin-4-ylmethyl)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4yl)cyclopropanamine;-   (1S,2R)-2-(4-(benzyloxy)phenyl)-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (1R,2S)-2-(4-(benzyloxy)phenyl)-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (1S,2R)—N-(2-(piperidin-4-yl)ethyl)-2-(pyridin-3yl)cyclopropanamine;-   (1R,2S)—N-(2-(piperidin-4-yl)ethyl)-2-(pyridin-3-yl)cyclopropanamine;-   (1S,2S)—N-(2-(piperidin-4-yl)ethyl)-2-(thiazol-5-yl)cyclopropanamine;-   (1R,2R)—N-(2-(piperidin-4-yl)ethyl)-2-(thiazol-5-yl)cyclopropanamine;-   (1S,2R)—N-(2-(piperidin-4-yl)ethyl)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4yl)cyclopropanamine;-   (1R,2S)—N-(2-(piperidin-4-yl)ethyl)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4yl)cyclopropanamine;-   (1S,2R)-2-(4-(benzyloxy)phenyl)-N-(2-(piperidin-4-yl)ethyl)cyclopropanamine;-   (1R,2S)-2-(4-(benzyloxy)phenyl)-N-(2-(piperidin-4-yl)ethyl)cyclopropanamine;-   (Trans)-2-phenyl-N-(pyrrolidin-3-ylmethyl)cyclopropanamine;-   (Trans)-2-(4-((2-fluorobenzyl)oxy)phenyl)-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (Trans)-N-(azetidin-3-ylmethyl)-2-phenylcyclopropanamine;-   (Trans)-2-(4-cyclopropylphenyl)-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (Trans)-N-(piperidin-4-ylmethyl)-2-(4-(pyridin-3-yl)phenyl)cyclopropanamine;-   (Trans)-2-(4-(1H-pyrazol-5-yl)phenyl)-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (Trans)-2-(naphthalen-2-yl)-N-(piperidin-4-ylmethyl)cyclopropanamine;-   2-methyl-2-phenyl-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (trans)-2-methyl-2-phenyl-N-(piperidin-4-ylmethyl)cyclopropanamine;-   (trans)-2-(4-(benzyloxy)phenyl)-N-((1-methylpiperidin-4-yl)methyl)cyclopropanamine;-   and pharmaceutically acceptable salts thereof.-   Still more preferably, the compound of formula (K) is a compound    from the list below:-   N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   N-((1S,2R)-2-phenylcyclopropyl)piperidin-4-amine;-   N-((1R,2S)-2-phenylcyclopropyl)piperidin-4-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropyl)tetrahydro-2H-pyran-4-amine;-   N-((trans)-2-(pyridin-3-yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(thiazol-5-yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-phenylcyclopropyl)piperidin-3-amine;-   N-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)piperidin-3-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)piperidin-3-amine;-   N-((trans)-2-phenylcyclopropyl)pyrrolidin-3-amine;-   N-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4yl)cyclopropyl)pyrrolidin-3-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)pyrrolidin-3-amine;-   N-((trans)-2-phenylcyclopropyl)azetidin-3-amine;-   N-((trans)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)azetidin-3-amine;-   N-((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)azetidin-3-amine;-   N-((trans)-2-phenylcyclopropyl)azepan-3-amine;-   N-((trans)-2-phenylcyclopropyl)-8-azabicyclo[3.2.1]octan-3-amine;-   N-((trans)-2-phenylcyclopropyl)-3-azabicyclo[3.2.1]octan-8-amine;-   N-((trans)-2-phenylcyclopropyl)decahydroquinolin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-1,2,3,4-tetrahydroquinolin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-3-azaspiro[5.5]undecan-9-amine;-   N-((trans)-2-phenylcyclopropyl)-2-azaspiro[4.5]decan-8-amine;-   N-((trans)-2-phenylcyclopropyl)-2,3-dihydrospiro[indene-1,4′-piperidin]-3-amine;-   N-((1S,2R)-2-(4-(benzyloxy)phenyl)cyclopropyl)piperidin-4-amine;-   N-((1R,2S)-2-(4-(benzyloxy)phenyl)cyclopropyl)piperidin-4-amine;-   N-((1S,2R)-2-(pyridin-3-yl)cyclopropyl)piperidin-4-amine;-   N-((1R,2S)-2-(pyridin-3-yl)cyclopropyl)piperidin-4-amine;-   N-((1S,2S)-2-(thiazol-5-yl)cyclopropyl)piperidin-4-amine;-   N-((1R,2R)-2-(thiazol-5-yl)cyclopropyl)piperidin-4-amine;-   N-((1S,2R)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)piperidin-4-amine;-   N-((1R,2S)-2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-7-azaspiro[3.5]nonan-2-amine;-   N-(2-(o-tolyl)cyclopropyl)piperidin-4-amine;-   N-(2-(2-fluorophenyl)cyclopropyl)piperidin-4-amine;-   N-(2-(3,4-difluorophenyl)cyclopropyl)piperidin 4 amine;-   N-(2-(4-methoxyphenyl)cyclopropyl)piperidin-4-amine;-   N-(2-(naphthalen-2-yl)cyclopropyl)piperidin-4-amine;-   N-(2-methyl-2-phenylcyclopropyl)piperidin-4-amine;-   N-(6-methoxy-4′-((trans)-2-(piperidin-4-ylamino)cyclopropyl)-[1,1′-biphenyl]-3-yl)methanesulfonamide;-   N-(4′-((trans)-2-(piperidin-4-ylamino)cyclopropyl)-[1,1′-biphenyl]-3-yl)propane-2-sulfonamide;-   1-(methylsulfonyl)-N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   1-(4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)piperidin-1-yl)ethanone;-   4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)piperidine-1-carboxamide;-   N-((trans)-2-(4-bromophenyl)cyclopropyl)tetrahydro-2H-pyran-4-amine;-   2,2,6,6-tetramethyl-N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   1-methyl-N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   1-isopropyl-N-((trans)-2-phenylcyclopropyl)piperidin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-1-(2,2,2-trifluoroethyl)piperidin-4-amine;-   N-((trans)-2-phenylcyclopropyl)-1-(pyridin-4-yl)piperidin-4-amine;-   4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)tetrahydro-2H-thiopyran    1,1-dioxide;-   N-((trans)-2-fluoro-2-phenylcyclopropyl)piperidin-4-amine;-   N-((1S,2S)-2-fluoro-2-phenylcyclopropyl)piperidin-4-amine;-   N-((1R,2R)-2-fluoro-2-phenylcyclopropyl)piperidin-4-amine;-   N-((trans)-2-(naphthalen-2-yl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-methyl-2-phenylcyclopropyl)piperidin-4-amine;-   N-((trans)-2-(o-tolyl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(2-fluorophenyl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(3,4-difluorophenyl)cyclopropyl)piperidin-4-amine;-   N-((trans)-2-(4-methoxyphenyl)cyclopropyl)piperidin-4-amine;

or a pharmaceutically acceptable salt thereof.

Compounds of formula (K) can be prepared by the methods disclosed inWO2013/057320, the disclosure of which is incorporated by referenceherein in its entirety.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (L) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

wherein

R₁ is selected from the group consisting of: C₁-C₆alkyl, —NSO₂Me,—NSO₂Ph, arylalkoxy, C₃-C₇cycloalkyl, —NC(O)R_(a),1-methyl-1H-pyrazol-4-yl, hydroxyl, C₁-C₄alkoxy, halogen, amide, amino,substituted amino, and —C(O)OR^(a);

R₂ is hydrogen or COOH;

each R₃ is independently selected from the group consisting of: aryl,heteroaryl, hydrogen, C₁-C₆alkyl, —SO₂R_(a), —NC(O)R_(a),—CH₂C(O)OR_(a), —C(O)OR_(a), —C(O)R_(a), —C(O)NR_(a)R_(b), substitutedamino, amino, urea, amide, sulfonamide, arylalkyl, and heteroarylalkyl;

each R_(a) is independently hydrogen, phenyl, phenylmethyl,3,5-dimethylisoxazol-4-yl, 1,2-dimethyl-1H-imidazol-4-yl,C₃-C₇cycloalkyl, C₁-C₆alkyl, C₁-C₄alkoxy, C₁-C₃alkylamino, or —NHPh;

R_(b) is hydrogen or C₁-C₃alkyl, or when attached to the same atom; or

R_(a) and R_(b) together form a 5- or 6-membered heterocycloalkyl ring;

R₄ is C₁-C₄alkyl, acyl, —C(O)CF₃ or hydrogen;

W is —(CH₂)₁₋₄, or —CH(R_(c))(CH₂)₀₋₃, in which R_(c) is CN orC₁-C₄alkyl;

Y is N or C;

X is N or C;

Z is O or (CH₂)_(q), wherein q is 0-2, when q is 0, Z represents a bond;

m is 0-3, n is 0-3;

provided that when Z is O, Y is N and X is C;

also provided that when X is C, at least one of the R₃ groups attachedto X is not hydrogen.

Compounds of formula (L) can be prepared by the methods disclosed inWO2012/135113, the disclosure of which is incorporated by referenceherein in its entirety.

Preferably, the compound of formula (L) is a compound from examples 1 to150 in WO2012/135113 or a pharmaceutically acceptable salt thereof.Still more preferably, the compound of formula (L) is4-((4-((((1R,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-1-yl)methyl)benzoicacid or a pharmaceutically acceptable salt thereof.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (M) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

A compound represented by the formula (M):

wherein A is a hydrocarbon group optionally having substituent(s), or aheterocyclic group optionally having substituent(s);

B is a benzene ring optionally having further substituent(s);

R¹, R² and R³ are each independently a hydrogen atom, a hydrocarbongroup optionally having substituent(s), or a heterocyclic groupoptionally having substituent(s);

A and R¹ are optionally bonded to each other to form, together with theadjacent nitrogen atom, a cyclic group optionally having substituent(s);and

R² and R³ are optionally bonded to each other to form, together with theadjacent nitrogen atom, a cyclic group optionally having substituent(s).

Compounds of formula (M) can be prepared by the methods disclosed inWO2014/058071, the disclosure of which is incorporated by referenceherein in its entirety.

Preferably, the compound of formula (M) is a compound from examples 1 to273 in WO2014/058071 or a pharmaceutically acceptable salt thereof. Morepreferably, the compound of formula (M) is3-(trans-2-((cyclopropylmethyl)amino)cyclopropyl)-N-(5-methyl-1,2-oxazol-3-yl)benzamide,3-(trans-2-((1-cyclopropylpiperidin-4-yl)amino)cyclopropyl)-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzamide,3-(trans-2-((cyclobutylamino)cyclopropyl)-N-(tetrahydro-2H-pyran-4-yl)benzamide,or a salt thereof.

In the methods and uses according to the invention, the KDM1A inhibitorcan be a compound of formula (N) or an enantiomer, a diastereomer or amixture of stereoisomers (such as a racemic mixture or a diastereomermixture) thereof, or a pharmaceutically acceptable salt or solvatethereof:

wherein A is a hydrocarbon group optionally having substituent(s), or aheterocyclic group optionally having substituent(s);

R is a hydrogen atom, a hydrocarbon group optionally havingsubstituent(s), or a heterocyclic group optionally havingsubstituent(s); or

A and R are optionally bonded to each other to form a ring optionallyhaving substituent(s);

Q¹, Q², Q³ and Q⁴ are each independently a hydrogen atom or asubstituent; Q¹ and Q², and Q³ and Q⁴, are each optionally bonded toeach other to form a ring optionally having substituent(s);

X is a hydrogen atom, an acyclic hydrocarbon group optionally havingsubstituent(s), or a saturated cyclic group optionally havingsubstituent(s);

Y¹, Y² and Y³ are each independently a hydrogen atom, a hydrocarbongroup optionally having substituent(s), or a heterocyclic groupoptionally having substituent(s);

X and Y¹, and Y¹ and Y², are each optionally bonded to each other toform a ring optionally having substituent(s); and

Z¹, Z² and Z³ are each independently a hydrogen atom or a substituent,

Compounds of formula (N) can be prepared by the methods disclosed inWO2013/022047, the disclosure of which is incorporated by referenceherein in its entirety.

Preferably, the compound of formula (N) is a compound from examples 1 to166 in WO2013/022047, or a pharmaceutically acceptable salt thereof.More preferably, the compound of formula (N) isN-(4-(trans-2-[(cyclopropylmethyl)amino]cyclopropyl)phenyl)biphenyl-4-carboxamide,N-(4-(trans-2-[(1-methylpiperidin-4-yl)amino]cyclopropyl)phenyl)-3-(trifluoromethyl)benzamide,N-(4-(trans-2-[(cyclopropylmethyl)amino]cyclopropyl)phenyl)-1H-pyrazole-4-carboxamide,or a salt thereof. A particularly preferred compound of formula (N) isN-[4-[2-[(cyclopropylmethylamino)methyl]cyclopropyl]phenyl]-1-methyl-pyrazole-4-carboxamide(Ex. 163), or a salt thereof.

The KDM1A inhibitor can also be the compound1-((4-(methoxymethyl)-4-(((1R,2S)-2-phenylcyclopropylamino)methyl)piperidin-1-yl)methyl)cyclobutanecarboxylicacid or a salt thereof, such as the p-toluenesulfonic acid salt. Thiscompound is disclosed in WO2017/27678.

A particularly preferred KDM1A inhibitor for use in the methods of theinvention is(trans)-N1-((1R,2S)-2-phenylcyclopropyl)cyclohexane-1,4-diamine, or asalt thereof.

Other preferred LSD1 inhibitors for use in the methods of the inventionare:

-   (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine;-   4-((4-((((1R,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-1-yl)methyl)benzoic    acid;

and salts thereof.

Preferably, the salts of KDM1A inhibitors as disclosed above arepharmaceutically acceptable salts. As used herein, a “pharmaceuticallyacceptable salt” is intended to mean a salt that retains the biologicaleffectiveness and/or properties of the parent compound (i.e. the freeacid or free base, as applicable) and that is not biologically orotherwise undesirable. Pharmaceutically acceptable salts include saltsformed with inorganic or organic bases, and salts formed with inorganicand organic acids. Pharmaceutically acceptable salts are well known inthe art. Exemplary pharmaceutically acceptable salts include those saltsprepared by reaction of the compounds of the present invention with amineral or organic acid, such as hydrochlorides, hydrobromides,sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrophosphates, dihydrophosphates, metaphosphates, pyrophosphates,chlorides, bromides, iodides, nitrates, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyne-1,4 dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates,citrates, lactates, gamma-hydroxybutyrates, glycollates, tartrates,methane-sulfonates, ethane-sulfonates, propanesulfonates,benzenesulfonates, toluenesulfonates, trifluoromethansulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates,pyruvates, stearates, ascorbates, or salicylates. When the compounds ofthe invention carry an acidic moiety, suitable pharmaceuticallyacceptable salts thereof may include alkali metal salts, e.g. sodium orpotassium salts; alkaline earth metal salts, e.g. calcium or magnesiumsalts; and salts formed with suitable organic ligands such as ammonia,alkylamines, hydroxyalkylamines, lysine, arginine, N-methylglucamine,procaine and the like. The pharmaceutically acceptable salts of thepresent invention can be prepared from the parent compound whichcontains a basic or acidic moiety by conventional chemical methods. Forexample, such salts can be prepared by reacting the free acid or baseforms of these compounds with a stoichiometric amount of the appropriatebase or acid in a suitable solvent.

It is to be understood that the present invention specifically relatesto each and every combination of features or embodiments describedherein, including any combination of general and/or preferredfeatures/embodiments. In particular, the invention specifically relatesto all combinations of preferred features/embodiments (including alldegrees of preference) of the methods and uses provided herein.

Unless otherwise stated, in the definitions of KDM1A inhibitors providedabove, particularly in the definitions of compounds of formula (A) to(N), the following definitions apply, when applicable:

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group cyclylC₁₋₈ alkyl wouldrepresent a cyclyl group attached to the parent molecule through a C₁₋₈alkyl group.

As used herein, the term “acyl” refers to a carbonyl attached to analkenyl, alkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, or any othermoiety where the atom attached to the carbonyl is carbon. Preferably,the term “acyl” refers to a group of formula —C(═O)R″, wherein R″represents alkenyl, alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl.An “acetyl” group refers to a —C(═O)CH₃ group. An “alkylcarbonyl” or“alkanoyl” group refers to an alkyl group attached to the parentmolecular moiety through a carbonyl group. Examples of such groupsinclude, but are not limited to, methylcarbonyl or ethylcarbonyl.Examples of acyl groups include, but are not limited to, formyl,alkanoyl or aroyl.

As used herein, the term “alkenyl” refers to a straight-chain orbranched-chain hydrocarbon group having one or more double bonds andcontaining from 2 to 20 carbon atoms. A C₂₋₈ alkenyl is an alkenyl grouphaving from 2 to 8 carbon atoms.

As used herein, the term “alkoxy” refers to an alkyl ether group (i.e. agroup of formula alkyl-O—), wherein the term alkyl is as defined below.Examples of suitable alkyl ether groups include, but are not limited to,methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy,sec-butoxy, tert-butoxy, or n-pentoxy. The term C_(1-z), alkoxy refersto an alkoxy group wherein the alkyl moiety has from 1 to z carbonatoms; for example a C₁₋₈ alkoxy is an alkoxy group wherein the alkylmoiety is C₁₋₈ alkyl, i.e. a group of formula C₁₋₈ alkyl-O—.

As used herein, the term “alkyl” refers to a straight-chain orbranched-chain alkyl group containing from 1 to 20 carbon atoms. AC_(1-z) alkyl is an alkyl from 1 to z carbon atoms; thus, a C₁₋₈ alkylhas from 1 to 8 carbon atoms, a C₁₋₄ alkyl has from 1 to 4 carbon atomsand a C₁₋₂ alkyl has from 1 to 2 carbon atoms. Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neo-pentyl,iso-amyl, hexyl, heptyl, octyl, or nonyl.

As used herein, the term “C₁₋₄ alkylene” refers to a C₁₋₄ alkyl groupattached at two positions, i.e. an alkanediyl group. Examples include,but are not limited to, methylene (i.e. a group of formula —CH₂—),ethylene (including ethane-1,2-diyl and ethane-1,1-diyl), propylene(e.g. propane-1,3-diyl, propane-1,2-diyl and propane-1,1-diyl) andbutylene (e.g. butane-1,4-diyl, butane-1,3-diyl or butane-1,1-diyl).Accordingly, the term “C₁₋₄ alkylene” may refer to a straight-chain orbranched-chain alkylene group having from 1 to 4 carbon atoms. A “linearC₁₋₄ alkylene” refers to a straight chain alkylene group having from 1to 4 carbon atoms, i.e. a —(CH₂)_(y)— group wherein y is 1, 2, 3 or 4.

As used herein, the term “alkylamino,” refers to an alkyl group attachedto the parent molecular moiety through an amino group. Suitablealkylamino groups may be mono- or dialkylated, forming groups including,but not limited to N-methylamino, N-ethylamino, N,N-dimethylamino,N,N-ethylmethylamino, N,N-diethylamino, N-propylamino, andN,N-methylpropylamino.

As used herein, the term “alkynyl” refers to a straight-chain orbranched-chain hydrocarbon group having one or more triple bonds andcontaining from 2 to 20 carbon atoms. A C₂₋₈ alkynyl has from 2 to 8carbon atoms. Examples of alkynyl groups include, but are not limitedto, ethynyl, propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl,pentyn-1-yl, 3-methylbutyn-1-yl, or hexyn-2-yl.

As used herein, the term “amido” and “carbamoyl” refers to an aminogroup as described below attached to the parent molecular moiety througha carbonyl group (e.g., —C(═O)NRR′), or vice versa (—N(R)C(═O)R′).“Amido” and “carbamoyl” encompasses “C-amido” and “N-amido” as definedherein. R and R′ are as defined herein.

As used herein, the term “C-amido” refers to a —C(═O)NRR′ group with Rand R′ as defined herein.

As used herein, the term “N-amido” refers to a —N(R)C(═O)R′ group with Rand R′ as defined herein.

As used herein, the term “amino” refers to —NRR′, wherein R and R′ areindependently selected from the group consisting of hydrogen, alkyl,heteroalkyl, aryl, carbocyclyl, and heterocyclyl. Additionally, R and R′may be combined to form a heterocyclyl. Exemplary “amino” groupsinclude, without being limited thereto, —NH₂, —NH(C₁₋₄ alkyl) and—N(C₁₋₄ alkyl)(C₁₋₄ alkyl).

As used herein, the term “aryl” refers to a carbocyclic aromatic systemcontaining one ring, or two or three rings fused together where in thering atoms are all carbon. The term “aryl” includes, but is not limitedto groups such as phenyl, naphthyl, or anthracenyl. The term “monocyclicaryl” refers to phenyl.

As used herein, the term “arylalkoxy” or “aralkoxy,” refers to an arylgroup attached to the parent molecular moiety through an alkoxy group.Examples of arylalkoxy groups include, but are not limited to, benzyloxyor phenethoxy.

As used herein, the term “arylalkyl” or “aralkyl,” refers to an arylgroup attached to the parent molecular moiety through an alkyl group.

As used herein, the term “aryloxy” refers to an aryl group attached tothe parent molecular moiety through an oxy (—O—).

As used herein, the term “carbamate” refers to an O-carbamyl orN-carbamyl group as defined herein. An N-carbamyl group refers to—NR—COOR′, wherein R and R′ are as defined herein. An O-carbamyl grouprefers to —OCO—NRR′, wherein R and R′ are as defined herein.

As used herein, the term “carbonyl” when alone includes formyl —C(═O)Hand in combination is a —C(═O)— group.

As used herein, the term “carboxyl” or “carboxy” refers to —C(═O)OH orthe corresponding “carboxylate” anion, such as is in a carboxylic acidsalt.

An “O-carboxy” group refers to a RC(═O)O— group, where R is as definedherein.

A “C-carboxy” group refers to a —C(═O)OR groups where R is as definedherein.

As used herein, the term “cyano” refers to —CN.

As used herein, the term “carbocyclyl” refers to a saturated orpartially saturated monocyclic or a fused bicyclic or tricyclic groupwherein the ring atoms of the cyclic system are all carbon and whereineach cyclic moiety contains from 3 to 12 carbon atom ring members.“Carbocyclyl” encompasses benzo fused to a carbocyclyl ring system. Onegroup of carbocyclyls have from 5 to 7 carbon atoms. Examples ofcarbocyclyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl,indanyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, or adamantyl.

As used herein, the term “cycloalkyl”, unless otherwise specified,refers to a saturated monocyclic, bicyclic or tricyclic group whereinthe ring atoms of the cyclic system are all carbon and wherein eachcyclic moiety contains from 3 to 12 carbon atom ring members. A C₃₋₆cycloalkyl is a cycloalkyl that has from 3 to 6 carbon atoms, i.e.cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. A cycloalkylcontaining from 4 to 7 C atoms includes cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl. Examples of cycloalkyl groups include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, or adamantyl.

As used herein, the term “cycloalkenyl” refers to a partially saturatedmonocyclic, bicyclic or tricyclic group wherein the ring atoms of thecyclic system are all carbon and wherein each cyclic moiety containsfrom 3 to 12 carbon atom ring members. One group of carboalkenyls hasfrom 5 to 7 carbon atoms. Examples of cycloalkenyl groups include, butare not limited to, cyclobutenyl, cyclopentenyl, or cyclohexenyl.

As used herein, the term “cyclyl” refers to an aryl, heterocyclyl, orcarbocyclyl group as defined herein.

As used herein, the term “cyclylC₁₋₈ alkyl” refers to a C₁₋₈ alkyl asdefined above wherein one hydrogen atom in the C₁₋₈ alkyl group has beenreplaced with one cyclyl group as defined above.

As used herein, the term “halo” or “halogen” refers to fluorine,chlorine, bromine, or iodine.

As used herein, the term “haloalkoxy” refers to a haloalkyl group (asdefined below) attached to the parent molecular moiety through an oxygenatom. A haloC₁₋₈ alkoxy group refers to a haloalkoxy group wherein thehaloalkyl moiety has from 1 to 8 C atoms. Examples of haloalkoxy groupsinclude, but are not limited to, trifluoromethoxy, 2-fluoroethoxy,pentafluoroethoxy, or 3-chloropropoxy.

As used herein, the term “haloalkyl” refers to an alkyl group having themeaning as defined above wherein one or more hydrogens are replaced witha halogen. A haloC₁₋₈ alkyl group refers to a haloalkyl group whereinthe alkyl moiety has from 1 to 8 C atoms. Specifically embraced aremonohaloalkyl, dihaloalkyl or polyhaloalkyl groups. A monohaloalkylgroup, for one example, may have an iodo, bromo, chloro or fluoro atomwithin the group. Dihalo or polyhaloalkyl groups may have two or more ofthe same halo atoms or a combination of different halo groups. Examplesof haloalkyl groups include, but are not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, chloromethyl, dichioromethyl,trichloromethyl, pentafluoroethyl, heptafluoropropyl,difluorochloromethyl, dichlorofluoromethyl, difluoroethyl,difluoropropyl, dichloroethyl or dichloropropyl.

As used herein, the term “heteroalkyl” refers to a straight or branchedalkyl chain, wherein one, two, or three carbons forming the alkyl chainare each replaced by a heteroatom independently selected from the groupconsisting of O, N, and S, and wherein the nitrogen and/or sulfurheteroatom(s) (if present) may optionally be oxidized and the nitrogenheteroatom(s) (if present) may optionally be quaternized. Theheteroatom(s) O, N and S may, for example, be placed at the end(s) or atan interior position of the heteroalkyl group, i.e., the heteroalkyl maybe bound to the remainder of the molecule via a heteroatom or a carbonatom. Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃. Accordingly, a further example for a “heteroalkyl” groupis a straight or branched alkyl group, in which two consecutive carbonatoms are replaced by the heteroatoms S and N, respectively, and thesulfur heteroatom is furthermore oxidized, resulting in moieties suchas, e.g., —S(═O)₂—NH₂, —S(═O)₂—NH(alkyl) or —S(═O)₂—N(alkyl)(alkyl).

As used herein, the term “heteroalkylene” refers to a heteroalkyl groupattached at two positions. Examples include, but are not limited to,—CH₂OCH₂—, —CH₂SCH₂—, and —CH₂NHCH₂—, —CH₂S—, or —CH₂NHCH(CH₃)CH₂—.

Accordingly, the term “heteroalkylene” may, e.g., refer to a straight orbranched alkylene group (i.e., a straight or branched alkanediyl group)having from 1 to 6 carbon atoms, wherein 1, 2 (if present) or 3 (ifpresent) of said carbon atoms are each replaced by a heteroatomindependently selected from O, N or S. It is to be understood that thepresence of hydrogen atoms will depend on the valence of the heteroatomreplacing the respective carbon atom. If, for example, the carbon atomin a —CH₂— group is replaced by O or S, the resulting group will be —O—or —S—, respectively, while it will be —N(H)— when the carbon atomreplaced by N. Likewise, if the central carbon atom in a group—CH₂—CH(—CH₃)—CH₂— is replaced by N, the resulting group will be—CH₂—N(—CH₃)—CH₂—.

An example for a “heteroalkylene” group is a straight or branchedalkylene group, in which two consecutive carbon atoms are replaced bythe heteroatoms S and N, respectively, and the sulfur heteroatom isfurthermore oxidized, resulting in moieties such as, e.g., —S(═O)₂—N(H)—or —S(═O)₂—N(alkyl)-. Accordingly, the groups —S(═O)₂—N(H)— and—S(═O)₂—N(alkyl)- (e.g., —S(═O)₂—N(C₁-C₅ alkyl)-) are exemplary“heteroalkylene” groups.

As used herein, the term “heteroC₁₋₄ alkylene” refers to a straight orbranched C₁₋₄ alkylene group (i.e., a straight or branched C₁₋₄alkanediyl group) linked to one heteroatom selected from O, N and S andalso refers to a straight or branched C₁₋₄ alkylene group wherein one ormore (e.g., 1, 2 (if present) or 3 (if present)) of the carbon atoms ofsaid alkylene group are each replaced by a heteroatom independentlyselected from O, N or S. The nitrogen and/or sulfur heteroatom(s) (ifpresent) may optionally be oxidized and the nitrogen heteroatom(s) (ifpresent) may optionally be quaternized. The heteroatom(s) O, N and S maybe placed at the end(s) and/or at an interior position of the heteroC₁₋₄alkylene group. It is to be understood that the presence of hydrogenatoms will depend on the valence of the heteroatom replacing therespective carbon atom. If, for example, the carbon atom in a —CH₂—group is replaced by O or S, the resulting group will be —O— or —S—,respectively, while it will be —N(H)— when the carbon atom is replacedby N. Likewise, if the central carbon atom in a group —CH₂—CH(—CH₃)—CH₂—is replaced by N, the resulting group will be —CH₂—N(—CH₃)—CH₂—. Anexample for a “heteroC₁₋₄ alkylene” group is a straight or branched C₁₋₄alkylene group, in which two consecutive carbon atoms are replaced bythe heteroatoms S and N, respectively, and the sulfur heteroatom isfurthermore oxidized, resulting in moieties such as, e.g., —S(═O)₂—N(H)—or —S(═O)₂—N(CH₃)—.

As used herein, the term “heteroaryl” refers to a 5 to 6 memberedunsaturated monocyclic ring, or a fused bicyclic or tricyclic ringsystem in which the rings are aromatic and in which at least one ringcontains at least one heteroatom selected from the group consisting ofO, S, and N. Preferred heteroaryl groups are 5- to 6-membered monocyclicor 9- to 10-membered bicyclic heteroaryl groups. Examples of heteroarylgroups include, but are not limited to, pyridinyl, imidazolyl,imidazopyridinyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl,tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl,oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl,benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,quinazolinyl, quinoxalinyl, naphthyridinyl, or furopyridinyl.

As used herein, the term “heterocyclyl” or “heterocycle” each refer to asaturated, partially unsaturated, or fully unsaturated monocyclic,bicyclic, or tricyclic heterocyclic group containing at least oneheteroatom as a ring member, wherein each said heteroatom may beindependently selected from the group consisting of nitrogen, oxygen,and sulfur wherein the nitrogen or sulfur atoms may be oxidized (e.g.,—N═O, —S(═O)—, or —S(═O)₂—). Additionally, 1, 2, or 3 of the carbonatoms of the heterocyclyl may be optionally oxidized (e.g., to give anoxo group or ═O). One group of heterocyclyls has from 1 to 4 heteroatomsas ring members. Another group of heterocyclyls has from 1 to 2heteroatoms as ring members. One group of heterocyclyls has from 3 to 8ring members in each ring. Yet another group of heterocyclyls has from 3to 7 ring members in each ring. Again another group of heterocyclyls hasfrom 5 to 6 ring members in each ring. “Heterocyclyl” is intended toencompass a heterocyclyl group fused to a carbocyclyl or benzo ringsystems. Examples of heterocyclyl groups include, but are not limitedto, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino,morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl,azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl,oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinylimidazolinyl, or imidazolidinyl. Examplesof heteroaryls that are heterocyclyls include, but are not limited to,pyridinyl, imidazolyl, imidazopyridinyl, pyrimidinyl, pyrazolyl,triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl,indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl,isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl,thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,naphthyridinyl, or furopyridinyl.

As used herein, the term “heterocycloalkyl” refers to a heterocyclylgroup that is not fully unsaturated e.g., one or more of the ringssystems of a heterocycloalkyl is not aromatic. Examples ofheterocycloalkyls include piperazinyl, morpholinyl, piperidinyl, orpyrrolidinyl.

As used herein, the term “hydroxyl” or “hydroxy” refers to —OH.

As used herein, the term “hydroxyalkyl,” as used herein, refers to ahydroxyl group attached to the parent molecular moiety through an alkylgroup.

As used herein, the term “hydroxyC₁₋₈ alkyl” refers to a C₁₋₈ alkylgroup, wherein one or more hydrogen atoms (preferably one or two) havebeen replaced by hydroxy groups.

As used herein, the term “R¹²R¹³N—C₁₋₈ alkyl” refers to a C₁₋₈ alkylgroup, wherein one or more hydrogen atoms (preferably one or two, morepreferably one) have been replaced by —NR¹²R¹³.

As used herein, the phrase “in the main chain,” refers to the longestcontiguous or adjacent chain of carbon atoms starting at the point ofattachment of a group to the compounds of any one of the formulasdisclosed herein.

As used herein, the term phrase “linear chain of atoms” refers to thelongest straight chain of atoms independently selected from carbon,nitrogen, oxygen and sulfur.

As used herein, the term “lower” where not otherwise specificallydefined, means containing from 1 to and including 6 carbon atoms.

As used herein, the term “lower aryl,” means phenyl or naphthyl.

As used herein, the term “nitro” refers to —NO₂.

As used herein, the term “saturated” in relation to a ring means thatthe ring does not contain any unsaturation.

As used herein, the terms “sulfonate” “sulfonic acid” and “sulfonic”refer to the —SO₃H group and its anion as the sulfonic acid is used insalt formation.

As used herein, the term “sulfanyl,” to —S—.

As used herein, the term “sulfinyl” refers to —S(═O)(R), with R asdefined herein.

As used herein, the term “sulfonyl” refers to —S(═O)₂R, with R asdefined herein.

As used herein, the term “sulfonamide” refers to an N-sulfonamido orS-sulfonamido group as defined herein.

As used herein, the term “N-sulfonamido” refers to a RS(═O)₂N(R′)— groupwith R and R′ as defined herein. Preferred N-sulfonamido groups are—NHSO₂R, wherein R is as defined herein, preferably R is alkyl,cycloalkyl, heteroalkyl, aryl, heteroaryl or heterocycloalkyl, morepreferably R is alkyl, aryl, heteroaryl or heterocycloalkyl, whereinsaid alkyl, said cycloalkyl, said heteroalkyl, said aryl, saidheteroaryl and said heterocycloalkyl are each optionally substituted.The optional substituents on said alkyl, said cycloalkyl, saidheteroalkyl, said aryl, said heteroaryl and said heterocycloalkyl may beselected independently from lower alkyl, lower alkenyl, lower alkynyl,lower alkanoyl, lower heteroalkyl, lower heterocycloalkyl, lowerhaloalkyl, lower cycloalkyl, phenyl, aryl, heteroaryl, pyridyl, aryloxy,lower alkoxy, lower haloalkoxy, oxo, lower acyloxy, carbonyl, carboxyl,lower alkylcarbonyl, lower carboxyester, lower carboxamido, cyano,halogen, hydroxyl, amino, amido, nitro, thiol, lower alkylthio, lowerhaloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonicacid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H,carbamate, and urea. Preferably, the optional substituents areindependently selected from hydroxyl, halo, alkyl, alkoxy, haloalkyl,haloalkoxy, —N(C₁₋₃ alkyl)₂, —NH(C₁₋₃ alkyl), —NHC(═O)(C₁₋₃ alkyl),—C(═O)OH, —C(═O)O(C₁₋₃ alkyl), —C(═O)(C₁₋₃ alkyl), —C(═O)NH₂,—C(═O)NH(C₁₋₃ alkyl), —C(═O)NH(cycloalkyl), —C(═O)N(C₁₋₃ alkyl)₂,—S(═O)₂(C₁₋₃ alkyl), —S(═O)₂NH₂, —S(═O)₂N(C₁₋₃ alkyl)₂, —S(═O)₂NH(C₁₋₃alkyl), —CHF₂, —OCF₃, —OCHF₂, —SCF₃, —CF₃, —CN, —NH₂, —NO₂, ortetrazolyl. Particularly preferred N-sulfonamido groups are —NHSO₂R,wherein R is alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl orheterocycloalkyl, and preferably R is alkyl, aryl, heteroaryl orheterocycloalkyl, and —NHSO₂ (optionally substituted aryl). Still morepreferred N-sulfonamido groups are —NHSO₂alkyl and —NHSO₂ (optionallysubstituted aryl). Exemplary, non-limiting N-sulfonamido groups are—NHSO₂alkyl such as —NHSO₂CH₃, —NHSO₂CH₂CH₃ or —NHSO₂(isopropyl), and—NHSO₂ (optionally substituted aryl) such as —NHSO₂-phenyl,—NHSO₂-(2-cyanophenyl), —NHSO₂-(3-cyanophenyl), —NHSO₂-(4-cyanophenyl),—NHSO₂-(2-aminophenyl), —NHSO₂-(3-aminophenyl) or—NHSO₂-(4-aminophenyl). Other exemplary N-sulfonamido groups are —NHSO₂(optionally substituted heterocycloalkyl) such as—NHSO₂-(piperazin-1-yl) and —NHSO₂ (optionally substituted heteroaryl)such as —NHSO₂-(optionally substituted pyridyl) like —NHSO₂-(3-pyridyl)or —NHSO₂-(6-amino-3-pyridyl).

As used herein, the term “S-sulfonamido” refers to a —S(═O)₂NRR′, group,with R and R′ as defined herein.

As used herein, the term “urea” refers to a —N(R)C(═O)N(R)(R′) groupwherein R and R′ are as defined herein.

As used herein, “hydrogen bonding group” refers to a substituent group,which is capable of taking part in a non-covalent bonding betweenhydrogen and another atom (usually nitrogen or oxygen). Examplesinclude, but are not limited to, —NH₂, —OH, amido, —S(O)₂NH₂, —C(═O)NH₂,—CH₂—C(═O)NH₂, - and —CH₂—NH₂. Other non-limiting examples includeNHC(═O)CH₃ or —NHCH₃.

As used herein, the term “amide isostere” refers to a monocyclic orbicyclic ring system that is isosteric or bioisosteric with an amidemoiety. Examples of amide isoteres include but are not limited to thosedisclosed in, e.g., Meanwell (2011) J. Med. Chem. PMID: 21413808,

The term R or the term R′, appearing by itself and without a numberdesignation, unless otherwise defined, refers to a moiety selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl,heteroaryl and heterocycloalkyl. Both unsubstituted and substitutedforms of the above groups are encompassed.

Whether an R group has a number designation or not, every R group,including R, R′ and R_(z) where z=(1, 2, 3, . . . z), every substituent,and every term should be understood to be independent of every other interms of selection from a group. Should any variable, substituent, orterm (e.g., aryl, heterocycle, R, etc.) occur more than one time in aformula or generic structure, its definition at each occurrence isindependent of the definition at every other occurrence. Those of skillin the art will further recognize that certain groups may be attached toa parent molecule or may occupy a position in a chain of elements fromeither end as written. Thus, by way of example only, an unsymmetricalgroup such as —C(═O)N(R)— may be attached to the parent moiety at eitherthe carbon or the nitrogen.

As used herein, the term “optionally substituted” means the preceding oranteceding group may be substituted or unsubstituted. When substitutedand unless otherwise specified, the substituents of an “optionallysubstituted” group may include, without limitation, one or moresubstituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower cycloalkyl, phenyl, aryl,heteroaryl, pyridyl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, halogen, hydroxyl, amino, amido, nitro, thiol,lower alkylthio, lower haloalkylthio, lower perhaloalkylthio, arylthio,sulfonate, sulfonic acid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃,CO₂CH₃, CO₂H, carbamate, and urea. Two substituents may be joinedtogether to form a fused five-, six-, or seven-membered carbocyclic orheterocyclic ring consisting of zero to three heteroatoms, for exampleforming methylenedioxy or ethylenedioxy. An optionally substituted groupmay be unsubstituted (e.g., —CH₂CH₃), fully substituted (e.g., —CF₂CF₃),monosubstituted (e.g., —CH₂CH₂F) or substituted at a level anywherein-between fully substituted and monosubstituted (e.g., —CH₂CF₃). Wheresubstituents are recited without qualification as to substitution, bothsubstituted and unsubstituted forms are encompassed. Where a substituentis qualified as “substituted,” the substituted form is specificallyintended. Additionally, different sets of optional substituents to aparticular moiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.” In one specific definition, the optionalsubstituents are chosen from hydroxyl, halo, alkyl, alkoxy, haloalkyl,haloalkoxy, —N(C₁₋₃ alkyl)₂, —NH(C₁₋₃ alkyl), —NHC(═O)(C₁₋₃ alkyl),—C(═O)OH, —C(═O)O(C₁₋₃ alkyl), —C(═O)(C₁₋₃ alkyl), —C(═O)NH₂,—C(═O)NH(C₁₋₃ alkyl), —C(═O)NH(cycloalkyl), —C(═O)N(C₁₋₃ alkyl)₂,—S(═O)₂(C₁₋₃ alkyl), —S(═O)₂NH₂, —S(═O)₂N(C₁₋₃alkyl)₂, —S(═O)₂NH(C₁₋₃alkyl), —CHF₂, —OCF₃, —OCHF₂, —SCF₃, —CF₃, —CN, —NH₂, —NO₂, ortetrazolyl.

As used herein, the term “optional substituent” denotes that thecorresponding substituent may be present or may be absent. Accordingly,a compound having 1, 2 or 3 optional substituents may be unsubstitutedor may be substituted with 1, 2 or 3 substituents, which may be the sameor different.

In accordance with the above, the present invention in particularrelates to:

-   -   1. A compound of formula (I)        P-L-Z  (I)        -   or a salt thereof,        -   wherein:        -   P is a tag or label;        -   L is a divalent C₆₋₁₀₀ hydrocarbon group, wherein one or            more carbon atoms comprised in said hydrocarbon group are            each optionally replaced by a heteroatom selected            independently from O, S and N, wherein one or more carbon            atoms comprised in said hydrocarbon group are each            optionally replaced by a group selected independently from            the group consisting of —C(═O)—, —NR¹—, —NR¹—C(═O)—,            —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,            —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,            —SO₂—NR¹— and —NR¹—SO₂—, and wherein L provides a distance            of at least 6 atoms between P and Z;        -   R¹ is hydrogen or C₁₋₄ alkyl; and        -   Z is a radical of a KDM1A inhibitor.    -   2. The compound of item 1, wherein Z is a radical of an        irreversible KDM1A inhibitor.    -   3. The compound of item 1 or 2, wherein Z is a radical of an        irreversible KDM1A inhibitor comprising a        2-cyclyl-cyclopropylamino moiety.    -   4. The compound of any one of items 1 to 3, wherein Z is a        radical of an irreversible KDM1A inhibitor disclosed in        WO2010/043721, WO2010/084160, WO2011/035941, WO2011/042217,        WO2011/131697, WO2012/013727, WO2012/013728, WO2012/045883,        WO2013/057320, WO2013/057322, WO2012/135113, WO2013/022047,        WO2014/058071, WO2010/143582, US2010/0324147, WO2011/131576,        WO2014/084298, WO2014/086790, WO2014/164867, WO2015/021128,        WO2015/123408, WO2015/123424, WO2015/123437, WO2015/123465,        WO2015/156417 or WO2015/181380.    -   5. The compound of any one of items 1 to 3, wherein Z is a group        of formula Z1

-   -   -   wherein:        -   A is aryl or heteroaryl, wherein said aryl or said            heteroaryl is optionally substituted;        -   B is aryl, heteroaryl or heterocycloalkyl, wherein B is            optionally substituted;        -   m is 0 or 1;        -   Y is a bond, —(C₁₋₄ alkylene)-O—, —(C₁₋₄ alkylene)-NR²—,            —(C₁₋₄ alkylene)-C(═O)—NR²—, or —(C₀₋₄ alkylene)-NR²—C(═O)—;        -   R² is hydrogen or C₁₋₄ alkyl;        -   D is hydrogen, —(C₁₋₄ alkylene)-CO—NR³R⁴, cyclyl or —(C₁₋₄            alkylene)-cyclyl, wherein cyclyl and the cyclyl moiety in            the —(C₁₋₄ alkylene)-cyclyl are each optionally substituted;        -   R³ and R⁴ are each independently selected from hydrogen,            C₁₋₄ alkyl and —(C₀₋₄ alkylene)-cyclyl, wherein the cyclyl            moiety in the —(C₀₋₄ alkylene)-cyclyl is optionally            substituted, or R³ and R⁴ are linked together to form            together with the N atom to which they are bound a            heterocyclic ring which may contain one or more additional            heteroatoms selected from N, O and S and which is optionally            substituted;        -   and the groups —(B—Y)_(m)-A- and —NH-D on the cyclopropyl            ring are in trans configuration.

    -   6. The compound of item 5, wherein D is —(C₁₋₄        alkylene)-CO—NR³R⁴, cyclyl or —(C₁₋₄ alkylene)-cyclyl, wherein        cyclyl and the cyclyl moiety in the —(C₁₋₄ alkylene)-cyclyl are        each optionally substituted.

    -   7. The compound of item 5, wherein D is cyclyl or —(C₁₋₄        alkylene)-cyclyl, wherein cyclyl and the cyclyl moiety in the        —(C₁₋₄ alkylene)-cyclyl are each optionally substituted.

    -   8. The compound of item 5, wherein D is cycloalkyl,        benzocycloalkyl, heterocycloalkyl or —(C₁₋₄ alkylene)-cyclyl,        wherein the cycloalkyl, the benzocycloalkyl, the        heterocycloalkyl and the cyclyl moiety in the —(C₁₋₄        alkylene)-cyclyl are each optionally substituted.

    -   9. The compound of item 5, wherein D is optionally substituted        cycloalkyl or optionally substituted benzocycloalkyl.

    -   10. The compound of item 9, wherein D is a group of formula

-   -   11. The compound of item 5, wherein D is optionally substituted        heterocycloalkyl.    -   12. The compound of item 11, wherein D is optionally substituted        piperidinyl.    -   13. The compound of item 11, wherein D is optionally substituted        4-piperidinyl.    -   14. The compound of item 5, wherein D is —(C₁₋₄ alkylene)-cyclyl        wherein the cyclyl moiety in the —(C₁₋₄ alkylene)-cyclyl is        optionally substituted.    -   15. The compound of item 14, wherein D is        —(C₁₋₄alkylene)-cycloalkyl wherein the cycloalkyl in the —(C₁₋₄        alkylene)-cycloalkyl is optionally substituted.    -   16. The compound of item 14, wherein D is —(C₁₋₄        alkylene)-heterocycloalkyl, wherein the heterocycloalkyl in the        —(C₁₋₄ alkylene)-heterocycloalkyl is optionally substituted.    -   17. The compound of item 16, wherein D is —CH₂-heterocycloalkyl,        preferably —CH₂-(4-piperidinyl), wherein the heterocycloalkyl in        the —CH₂-heterocycloalkyl and the 4-piperidinyl in the        —CH₂-(4-piperidinyl) are each optionally substituted.    -   18. The compound of item 17, wherein D is a group of formula

-   -   19. The compound of item 14, wherein D is —(C₁₋₄        alkylene)-heteroaryl, preferably —CH₂-heteroaryl, wherein the        heteroaryl in the —(C₁₋₄ alkylene)-heteroaryl and the heteroaryl        in the —CH₂-heteroaryl is optionally substituted.    -   20. The compound of item 19, wherein D is a group of formula

-   -   21. The compound of any of items 5 to 20, wherein Y is a bond,        —CH₂—O—, —C(═O)—NR²—, or —NR²—C(═O)—.    -   22. The compound of any of items 5 to 21, wherein R² is        hydrogen.    -   23. The compound of any of items 5 to 22, wherein A is        optionally substituted phenyl.    -   24. The compound of any of items 5 to 22, wherein A is phenyl.    -   25. The compound of any of items 5 to 24, wherein m is 0.    -   26. The compound of any of items 5 to 24, wherein m is 1.    -   27. The compound of item 26, wherein Y is a bond.    -   28. The compound of any of items 5 to 24 or 26 to 27, wherein B        is aryl or heteroaryl, wherein B is optionally substituted.    -   29. The compound of any of items 5 to 24 or 26 to 28, wherein B        is optionally substituted aryl, preferably optionally        substituted phenyl.    -   30. The compound of item 29, wherein B is phenyl.    -   31. The compound of any one of items 1 to 3 or 5, wherein Z is a        group of formula Z2

-   -   wherein the phenyl ring is optionally substituted, and wherein        the substituents on the cyclopropyl ring are in trans        configuration.    -   32. The compound of item 31, wherein Z is a group of formula Z3a        or Z3b

-   -   wherein in Z3a and Z3b the phenyl ring is optionally substituted        and the substituents on the cyclopropyl ring are in trans        configuration    -   33. The compound of item 31, wherein Z is a group of formula Z4:

-   -   34. The compound of item 33, wherein Z is a group of formula Z5a        or Z5b:

-   -   35. The compound of any one of items 1 to 25, wherein L provides        a distance between P and Z of 6 to 90 atoms.    -   36. The compound of any one of items 1 to 35, wherein L is a        divalent C₃₀-C₆₀ hydrocarbon group, wherein one or more carbon        atoms comprised in said hydrocarbon group are each optionally        replaced by a heteroatom selected independently from O, S and N,        wherein one or more carbon atoms comprised in said hydrocarbon        group are each optionally replaced by a group selected        independently from the group consisting of —C(═O)—, —NR¹—,        —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,        —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—, —SO₂—NR¹—        and —NR¹—SO₂—, and wherein L provides a distance between P and Z        of 25 to 70 atoms.    -   37. The compound of any one of items 1 to 36, wherein L is a        divalent C₄₀-C₇₀ hydrocarbon group, wherein one or more carbon        atoms comprised in said hydrocarbon group are each optionally        replaced by a heteroatom selected independently from O, S and N,        wherein one or more carbon atoms comprised in said hydrocarbon        group are each optionally replaced by a group selected        independently from the group consisting of —C(═O)—, —NR¹—,        —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,        —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—, —SO₂—NR¹—        and —NR¹—SO₂—, and wherein L provides a distance between P and Z        of 35 to 65 atoms.    -   38. The compound of any one of items 1 to 35, wherein L        comprises a heteroalkylene group of 6 to 70 atoms, preferably of        6 to 50 atoms.    -   39. The compound of any one of items 1 to 35 wherein L comprises        a group of formula (i) or (ii):        -   (i) —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is an integer from            6 to 20, preferably from 8 to 18, and p is an integer from 1            to 5; or    -   (ii) —(CH₂CH₂O)_(x)—(CH₂)_(q)-G-(CH₂CH₂O)_(y)(CH₂)_(r)—, wherein        G is —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,        —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, or —O—C(═O)—,        preferably G is —NR¹—C(═O)— or —C(═O)—NR¹—, one of x and y is an        integer from 1 to 18 and the other from x and y is an integer        from 0 to 17 provided that x+y is from 4 to 18, preferably x+y        is from 6 to 16, and each of q and r is an integer independently        selected from 1 to 5.    -   40. The compound of item 39, wherein L comprises a group of        formula (i) or (ii):        -   (i) —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is an integer from            6 to 20, preferably from 8 to 18, and p is an integer from 1            to 5; or        -   (ii) —(CH₂CH₂O)_(x)—(CH₂)_(q)-G-(CH₂CH₂O)_(y)(CH₂)_(r)—,            wherein G is —NR¹—C(═O)— or —C(═O)—NR¹—, one of x and y is            an integer from 1 to 18 and the other from x and y is an            integer from 0 to 17 provided that x+y is from 4 to 18,            preferably x+y is from 6 to 16, and each of q and r is an            integer independently selected from 1 to 5.    -   41. The compound of any one of items 1 to 35 wherein L comprises        a group of formula —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is an        integer from 6 to 20, preferably from 8 to 18, and p is an        integer from 1 to 5.    -   42. The compound of any of of items 1 to 41, wherein L comprises        a group of formula X₁—X₂—X₃, wherein X₁ is linked to the        remainder of L and X₃ is linked to Z, and wherein:        -   X₁ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—,            —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—,            —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—, —SO₂—NR¹—, —NR¹—SO₂— or            —O—;        -   X₂ is C₀₋₅ alkylene;        -   X₃ is arylene or heteroarylene, wherein said arylene and            said heteroarylene are each optionally substituted.    -   43. The compound of item 42, wherein X₁ is —NHC(═O)—, —C(═O)NH—,        —SO₂—NH— or —NH—SO₂—.    -   44. The compound of item 42 or 43, wherein X₁ is —NHC(═O)— or        —C(═O)NH—, preferably —NHC(═O)—.    -   45. The compound of any of items 42 to 44, wherein X₃ is        connected to X₁-X₂ and to Z in a 1,3 disposition.    -   46. The compound of item 45, wherein X₃ is a group of formula:

-   -   -   wherein each ring is optionally substituted.

    -   47. The compound of item 46, wherein X₃ is a group of formula:

which is optionally substituted.

-   -   48. The compound of any one of items 42 to 47, wherein X₂ is        C₁₋₅ alkylene, preferably —(CH₂)₁₋₅—.    -   49. The compound of item 42, wherein X₁ is —NHC(═O)— or        —C(═O)NH—, X₂ is C₁₋₅ alkylene and X₃ is a group of formula:

-   -   50. The compound of item 42, wherein X₁—X₂—X₃ is a group of        formula:

-   -   -   wherein the group is linked to Z through the phenyl ring and            to the remainder of L through the N atom.        -   A compound of formula (II)

-   -   -   or a salt thereof,        -   wherein:        -   P is a tag or label;        -   D is cyclyl or —(C₁₋₄ alkylene)-cyclyl, wherein cyclyl and            the cyclyl moiety in the —(C₁₋₄ alkylene)-cyclyl are each            optionally substituted;        -   L₁ is a divalent C₆₋₉₀ hydrocarbon group, wherein one or            more carbon atoms comprised in said hydrocarbon group are            each optionally replaced by a heteroatom selected            independently from O, S and N, wherein one or more carbon            atoms comprised in said hydrocarbon group are each            optionally replaced by a group selected independently from            the group consisting of —C(═O)—, —NR¹—, —NR¹—C(═O)—,            —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,            —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,            —SO₂—NR¹— and —NR¹—SO₂—, and wherein L₁ provides a distance            of at least 3 atoms between P and X₁;        -   R¹ is hydrogen or C₁₋₄ alkyl;        -   X₁ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—,            —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—,            —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—, —SO₂—NR¹—, —NR¹—SO₂— or            —O—;        -   X₂ is C₀₋₅ alkylene;        -   one of Z₁ and Z₂ is CH or N, and the other of Z₁ and Z₂ is            CH;        -   s and t are each independently selected from 0, 1 and 2;        -   R₅ and R₆ are at each occurrence independently selected from            C₁₋₄ alkyl, halo, —NH₂, —NR^(a)R^(c), —CN, —OH, —OR^(c),            haloC₁₋₄alkyl, cyclyl, cyclylC₁₋₄ alkyl-, C₁₋₄ alkyl-O—C₁₋₄            alkyl;        -   R^(a) is selected from hydrogen, C₁₋₄ alkyl and haloC₁₋₄            alkyl;        -   R_(c) is independently selected from C₁₋₄ alkyl, haloC₁₋₄            alkyl, cyclyl, cyclylC₁₋₄ alkyl-, and C₁₋₄ alkyl-O—C₁₋₄            alkyl-; and        -   wherein the phenyl and —NH-D groups on the cyclopropyl ring            are in trans configuration.

    -   52. The compound of item 51, which has formula (IIa), or a salt        thereof

-   -   53. The compound of item 51 or 52 wherein D is cycloalkyl,        benzocycloalkyl, heterocycloalkyl or —(C₁₋₄ alkylene)-cyclyl,        wherein the cycloalkyl, the benzocycloalkyl, the        heterocycloalkyl and the cyclyl moiety in the —(C₁₋₄        alkylene)-cyclyl are each optionally substituted.    -   54. The compound of item 51 or 52 wherein D is optionally        substituted cycloalkyl or optionally substituted        benzocycloalkyl.    -   55. The compound of item 51 or 52 wherein D is optionally        substituted heterocycloalkyl    -   56. The compound of item 51 or 52, wherein D is optionally        substituted piperidinyl    -   57. The compound of item 51 or 52, wherein D is optionally        substituted 4-piperidinyl    -   58. The compound of item 51 or 52 wherein D is —(C₁₋₄        alkylene)-cyclyl wherein the cyclyl moiety in the —(C₁₋₄        alkylene)-cyclyl is optionally substituted.    -   59. The compound of item 51 or 52 wherein D is        —(C₁₋₄alkylene)-cycloalkyl wherein the cycloalkyl in the —(C₁₋₄        alkylene)-cycloalkyl is optionally substituted.    -   60. The compound of item 51 or 52 wherein D is —(C₁₋₄        alkylene)-heterocycloalkyl, wherein the heterocycloalkyl in the        —(C₁₋₄ alkylene)-heterocycloalkyl is optionally substituted.    -   61. The compound of item 51 or 52 wherein D is        —CH₂-heterocycloalkyl, preferably —CH₂-(4-piperidinyl), wherein        the heterocycloalkyl in the —CH₂-heterocycloalkyl and the        4-piperidinyl in the —CH₂-(4-piperidinyl) are each optionally        substituted.    -   62. The compound of item 51 or 52 wherein D is —(C₁₋₄        alkylene)-heteroaryl, preferably —CH₂-heteroaryl, wherein the        heteroaryl in the —(C₁₋₄ alkylene)-heteroaryl and the heteroaryl        in the —CH₂-heteroaryl is optionally substituted.

63. The compound of item 51 or 52 wherein D is a group of formula

-   -   64, The compound of item 51, which has formula (III), or a salt        thereof

-   -   -   wherein the substituents on the cyclopropyl ring are in            trans configuration.

    -   65. The compound of item 64, which has formula (IIIa), or a salt        thereof

-   -   66. The compound of any of items 51 to 65, wherein X, is        —NR¹—C(═O)—, —C(═O)—NR¹—, —SO₂—NR¹— or —NR¹—SO₂—.    -   67. The compound of item 66 wherein X, is —NHC(═O)—, —C(═O)NH—,        —SO₂—NH— or —NH—SO₂—.    -   68. The compound of item 66 wherein X, is —NHC(═O)— or        —C(═O)NH—.    -   69. The compound of item 66 wherein X₁ is —NHC(═O)—.    -   70. The compound of any of items 51 to 69, wherein X₂ is C₁₋₅        alkylene, preferably —(CH2)₁₋₅—.    -   71. The compound of any one of items 51 to 65 wherein X₁-X₂ is a        group of formula

wherein the group is linked to the ring through the carbon atom and toL1 through the N atom.

-   -   72. The compound of any of items 51 to 71, wherein each of Z₁        and Z₂ is CH.    -   73. The compound of any of items 51 to 72, wherein each of s and        t is 0.    -   74. The compound of any of items 51 to 73, wherein L₁ is a group        of formula X₄-X₅, wherein X₄ is linked to P and X₅ is linked to        X₁, wherein:        -   X₄ is —C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—,            —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—,            —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—, —SO₂—NR¹— or —NR¹—SO₂—;            and        -   X₅ is a group of formula (i) or (ii):        -   (i) —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is an integer from            6 to 20, preferably from 8 to 18, and p is an integer from 1            to 5; or        -   (ii) —(CH₂CH₂O)_(x)—(CH₂)_(q)-G-(CH₂CH₂O)_(y)(CH₂)_(r)—,            wherein G is —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,            —NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, or            —O—C(═O)—, preferably G is —NR¹—C(═O)— or —C(═O)—NR¹—, one            of x and y is an integer from 1 to 18 and the other from x            and y is an integer from 0 to 17 provided that x+y is from 4            to 18, preferably x+y is from 6 to 16, and each of q and r            is an integer independently selected from 1 to 5.    -   75. The compound of item 74, wherein X₅ is a group of        formula (i) or (ii):        -   (i) —(CH₂CH₂O)_(n)—(CH2)_(p)—, wherein n is an integer from            6 to 20, preferably from 8 to 18, and p is an integer from 1            to 5; or        -   (ii) —(CH₂CH₂O)_(x)—(CH₂)_(q)-G-(CH₂CH₂O)_(y)(CH₂)_(r)—,            wherein G is —NR¹—C(═O)— or —C(═O)—NR¹—, one of x and y is            an integer from 1 to 18 and the other from x and y is an            integer from 0 to 17 provided that x+y is from 4 to 18,            preferably x+y is from 6 to 16, and each of q and r is an            integer independently selected from 1 to 5.    -   76. The compound of item 74, wherein X5 is a group of formula        —(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is an integer from 6 to 20,        preferably from 8 to 18, and p is an integer from 1 to 5.    -   77. The compound of any one of items 1 to 76, wherein P is a        tag.    -   78. The compound of item 77, wherein P is biotin or a biotin        derivative.    -   79. The compound of any one of items 1 to 76, wherein P is a        label.    -   80. The compound of item 79, wherein P is a fluorescent label.    -   81. The chemoprobe of item 80, wherein P is fluorescein or a        fluorescein derivative.    -   82. The compound of any one of items 1 to 81, wherein the        compound has an IC50 against KDM1A below 1 mcM, more preferably        below 500 nM.    -   83. The compound of any one of items 1 to 82, with a selectivity        of at least 10-fold for KDM1A over other FAD-dependent monoamine        oxidases.    -   84. The compound of any one of items 1 to 82, with a selectivity        of at least 30-fold for KDM1A over other FAD-dependent monoamine        oxidases.    -   85. The compound of any one of items 1 to 82 with a selectivity        of at least 50-fold for KDM1A over other FAD-dependent monoamine        oxidases.    -   86. The compound of item 1 or 51 selected from

and salts thereof.

-   -   87. The compound of item 1 or 51 selected from

-   -   -   and salts thereof.

    -   88. The compound of item 1 or 51, wherein the compound is

-   -   -   or a salt thereof.

    -   89. A method for determining a level of free KDM1A in a sample        or subject, wherein said method comprises        -   (i) contacting or exposing KDM1A to a chemoprobe, wherein            said chemoprobe is a compound of any one of items 1 to 88;            and        -   (ii) determining said level of free KDM1A employing said            chemoprobe in said sample or subject.

    -   90. A method for in vitro determining a level of free KDM1A in a        sample, wherein said method comprises        -   (i) contacting or exposing KDM1A to a chemoprobe, wherein            said chemoprobe is a compound of any one of items 1 to 88;            and        -   (ii) determining said level of free KDM1A employing said            chemoprobe in said sample.

    -   91. The method of item 89 or 90, wherein said method further        comprises to determine a level of total KDM1A in said sample.

    -   92. The method of any one of items 89 to 91, wherein said method        further comprises determining target engagement of a KDM1A        inhibitor in said sample.

    -   93. The method of item 92, wherein the determination of said        target engagement comprises calculating the ratio between free        KDM1A level in the sample and the free KDM1A level in a        reference sample.

    -   94. The method of item 92, wherein the determination of said        target engagement comprises calculating the ratio between free        KDM1A level and total KDM1A level in the sample.

    -   95. The method of item 92, wherein the determination of said        target engagement comprises calculating the ratio R_(X)/R_(REF),        wherein R_(X) is the ratio of the free KDM1A level and the total        KDM1A level in the sample and R_(REF) is the ratio of the free        KDM1A level and total KDM1A level in the reference sample.

    -   96. The method of item 92, wherein the target engagement is        determined as 1 minus the ratio calculated according to any of        items 93 to 95, wherein 1 corresponds to full target engagement        and 0 corresponds to absence of target engagement.

    -   97. A method for in vitro determining target engagement of an        inhibitor of KDM1A in a sample, wherein said method comprises        -   (i) contacting or exposing KDM1A to a chemoprobe, wherein            said chemoprobe is a compound of any one of items 1 to 88;        -   (ii) determining a level of free KDM1A employing said            chemoprobe in said sample;        -   (iii) determining a level of free KDM1A in a reference            sample;        -   (iv) calculating the ratio between the free KDM1A level in            the sample and the free KDM1A level in the reference sample;            and        -   (v) determining target engagement as 1 minus the ratio            calculated in step (iv).

    -   98. A method for in vitro determining target engagement of an        inhibitor of KDM1A in a sample, wherein said method comprises        -   (i) contacting or exposing KDM1A to a chemoprobe, wherein            said chemoprobe is a compound of any one of items 1 to 88;        -   (ii) determining a level of free KDM1A employing said            chemoprobe in said sample        -   (iii) determining a level of total KDM1A in said sample;        -   (iv) calculating the ratio between free KDM1A level and            total KDM1A level in the sample; and        -   (v) determining target engagement as 1 minus the ratio            calculated in step (iv).

    -   99. A method for in vitro determining target engagement of an        inhibitor of KDM1A in a sample, wherein said method comprises        -   (i) contacting or exposing KDM1A to a chemoprobe, wherein            said chemoprobe is a compound of any one of items 1 to 88;        -   (ii) determining a level of free KDM1A employing said            chemoprobe in said sample;        -   (iii) determining a level of total KDM1A in said sample;        -   (iv) determining a level of free KDM1A employing said            chemoprobe in a reference sample;        -   (v) determining a level of total KDM1A in said reference            sample;        -   (vi) calculating the ratio A/B, wherein A is the ratio of            the free KDM1A level and the total KDM1A level in the sample            and B is the ratio of the free KDM1A level and the total            KDM1A level in the reference sample; and        -   (vii) determining target engagement as 1 minus the ratio            calculated in step (vi).

    -   100. The method according to any one of items 97 to 99, wherein        said method is used to assess pharmacodynamics of KDM1A target        engagement.

    -   101. A method for in vitro determining a spatial distribution of        free KDM1A in a sample, wherein said method comprises        -   (i) contacting or exposing KDM1A to a chemoprobe, wherein            said chemoprobe is a compound of any one of items 1 to 88;            and        -   (ii) visualizing the spatial distribution of said free KDM1A            in said sample by detection of the chemoprobe.

    -   102. The method of item 101, wherein said method further        comprises determining the spatial distribution of total KDM1A in        said sample.

    -   103. A method for in vitro determining interaction factors of        KDM1A,        -   wherein said method comprises        -   (i) contacting or exposing a sample to a chemoprobe, wherein            said chemoprobe is a compound of any one of items 1 to 88;        -   (ii) isolating chemoprobe-bound KDM1A-containing complexes;        -   (iii) identifying said interaction factors of KDM1A, wherein            said interaction factors are nucleic acid(s) and/or            polypeptide(s).

    -   104. The method of any one of items 89 to 103, wherein said        sample is obtained from a subject.

    -   105. The method of item 104, wherein said subject has been        administered a KDM1A inhibitor.

    -   106. The method of any one of items 89 to 103, wherein said        sample has been exposed or contacted to a KDM1A inhibitor.

    -   107. The method of any one of items 92 to 100, wherein said        inhibitor of KDM1A is an irreversible inhibitor of KDM1A.

    -   108. The method of item 107, wherein said irreversible KDM1A        inhibitor is a compound disclosed in: WO2010/043721,        WO2010/084160, WO2011/035941, WO2011/042217, WO2011/131697,        WO2012/013727, WO2012/013728, WO2012/045883, WO2013/057320,        WO2013/057322, WO2010/143582, US2010-0324147, WO2011/131576,        WO2012/135113, WO2013/022047, WO2014/058071, WO2014/084298,        WO2014/086790, WO2014/164867, WO2015/021128; WO2015/123408,        WO2015/123424, WO2015/123437, WO2015/123465, WO2015/156417 or        WO2015/181380

    -   109. The method of item 108, wherein said irreversible KDM1A        inhibitor is        (trans)-N1-((1R,2S)-2-phenylcyclopropyl)cyclohexane-1,4-diamine,        (−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine,        or        4-((4-((((1R,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-1-yl)methyl)benzoic        acid, or a salt thereof.

    -   110. The method of any one of items 92 to 100, wherein said        inhibitor of KDM1A is a reversible inhibitor of KDM1A.

    -   111. The method of item 110, wherein said reversible inhibitor        of KDM1A is a compound disclosed in WO2007/021839,        WO2008/127734, WO2011/022489, WO2012/034116, WO2012/071469,        WO2013/025805, US2015/0065434, WO2013/033688, CN103054869,        CN103319466, WO2014/085613, CN103893163A, CN103961340,        WO2014/205213, WO2015/031564, WO2015/089192, WO2015/120281,        WO2015/134973, WO2015/168466, WO2015/200843, WO2016/003917,        WO2016/004105, WO2016/007722, WO2016/007727, WO2016/007731,        WO2016/007736, WO2016/034946 and WO2016/037005.

    -   112. The method of any one of items 89 to 111, wherein the        chemoprobe is used to capture or detect free KDM1A.

    -   113. The method of 112, wherein P in the chemoprobe is a tag and        the chemoprobe is captured or detected by a suitable capture or        detection agent.

    -   114. The method of item 112 or 113, wherein the determination of        the level of free KDM1A comprises the use of an antibody        specific to an epitope of KDM1A.

    -   115. The method of any one of items 112 to 114,

    -   wherein the determination of the level of total KDM1A comprises        the use of a first antibody specific to an epitope of KDM1A to        capture or detect total KDM1A.

    -   116. The method of item 115,

    -   wherein the determination of the level of total KDM1A comprises        the use of a second antibody specific to an epitope of KDM1A to        capture or detect total KDM1A, wherein said epitope of said        second antibody is different from said epitope of the first        antibody.

    -   117. The method of any one of item 89 to 116 which comprises the        use of a protein to capture or detect the tag in the chemoprobe        and to determine said level of free KDM1A

    -   118. The method of item 116, wherein one of said antibodies is        directed against an epitope located at the N-terminal region of        KDM1A and the other antibody is directed against an epitope        located at the the C-terminal region of KDM1A.

    -   119. The method of item 116, wherein one of said antibodies        targets epitope EP1, wherein EP1 is located in the N terminal        region proximate to proline 60 (P60) of the human KDM1A sequence        (UNIPROT ID O60341-1) and is blocked by peptide #LSD1 Blocking        Peptide-2184 specific (Cell Signaling).

    -   120. The method of item 119, wherein the other of said        antibodies targets epitope EP2, wherein EP2 is located on the        C-terminal region and comprises AMYTLPRQATPGVPAQ, corresponding        to AA 832-847 of human KDM1A.

    -   121. The method of item 120, wherein the antibodies are mAb-844        and mAb-825, the tag P in the KDM1A chemoprobe is biotin and the        protein used to capture the chemoprobe is streptavidin.

    -   122. The method of item 121, wherein the level of total KDM1A is        determined by sandwich ELISA and the level of free KDM1A is        determined by a KDM1A chemoprobe capture ELISA.

    -   123. The method of item 122, where the ELISA uses        chemoluminescent detection.

    -   124. The method of item 101 or 102, where said method further        comprises determining the spatial distribution of a downstream        KDM1A target in said sample.

    -   125. The method of item 103, wherein said interaction factors of        KDM1A are identified by mass spectrometry, antibody microarray        or Western Blot analysis.

    -   126. The method of item 103, wherein said interaction factors of        KDM1A are identified identified by qPCR, DNA microarray analysis        or next generation sequencing.

The following examples illustrate various aspects of the invention. Theexamples should, of course, be understood to be merely illustrative ofonly certain embodiments of the invention and not to constitutelimitations upon the scope of the invention.

EXAMPLES

Unless stated otherwise, in the compounds of all Examples of the presentspecification the stereochemical configuration is defined by thechemical name indicated for the respective compound, even though thedrawn structure may represent a more specific configuration.Nevertheless, the invention relates to all stereoisomers of thecompounds described and defined herein. Accordingly, the inventionencompasses the compounds described in the Examples as defined by theirchemical names and, in addition thereto, also the correspondingcompounds having the absolute configuration shown in the respectivedrawn structures.

The following abbreviations have been used:

ACN: acetonitrile, AcOH: acetic acid, aq: aqueous, Boc:tert-butyloxycarbonyl, (Boc)₂O: di-tert-butyl dicarbonate, DCE:1,2-dichloroethane, DCM: dichloromethane, DMF: N,N-dimethylformamide,DMSO: dimethylsulfoxide, DIPEA: N,N-Diisopropylethylamine, Et₂O: diethylether, EtOAc: ethyl acetate, HATU:1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, HPLC: high performance liquidchromatography, LC-MS: Liquid chromatography-mass spectrometry, m:multiplet, MeOH: methanol, NMR: nuclear magnetic resonance, Pd(PPh₃)₄:Tetrakis(triphenylphosphine)palladium(0), PEG: Polyethylene glycol, Petether: petroleum ether, q: quadruplet, Rt: retention time, RT: roomtemperature, sat.: saturated, TE: target engagement, TEA: triethylamine,THF: tetrahydrofuran, TLC: thin layer chromatography, urn: micrometers,UPLC: Ultra Performance Liquid Chromatography.

As used herein, μ, u, mc have been used interchangeably to indicatemicro, as in μM, uM, mcM: micromolar, μg, ug, mcg: microgram, μl, ul:microliter, μm, urn: micrometer, etc.

Example 1 Synthesis of Chemoprobes

One of the following methods was used for the determination by LC-MS:

Method 1: Column: Xbridge C18 (100 mm×4.6 mm, 3.5 um); Mobile Phase: A:Acetonitrile, B: 0.01 M Ammonium Bicarbonate in Water; Gradient: Time/%A: 0/5, 315, 10/80, 12/90, 15/90, 15.1/5; Flow Rate: 1.0 mL/min;Diluent: Acetonitrile:Water (70:30)

Method 2: Column: Aquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 um); MobilePhase: A: 0.05% Formic Acid in Water B: 0.05% Formic Acid inAcetonitrile; Gradient: Time/% A: 0/97, 0.3/97, 3.2/2, 3.8/2, 4.3/97,4.5/97; Column Temp: 35° C.; Flow Rate: 0.6 mL/min

Method 3: Column: Aquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 um); MobilePhase: A: 0.1% Formic Acid in Water B: 0.1% Formic Acid in Acetonitrile;Gradient: Time/% B: 0/3, 0.4/3, 3.2/98, 3.8/98, 4.2/3, 4.5/3; ColumnTemp: 35° C.; Flow Rate: 0.6 mL/min

Method 4: Column: Xbridge C18 (100 mm×4.6 mm, 3.5 um); Mobile Phase: A:Acetonitrile, B: 0.01 M Ammonium Bicarbonate in Water; Gradient: Time/%B: 0/90, 3/15, 5/2, 7/2, 8/90, 10/90; Flow Rate: 1.0 mL/min; Diluent:Water

Method 5: Column: Xbridge C18 (100 mm×4.6 mm, 3.5 um); Mobile Phase: A:100% Acetonitrile, B: Water; Gradient: Time/% B: 0/100, 5/100, 8/10,10/10, 15/98; Flow Rate: 1.0 mL/min; Diluent: Water

Method 6: Column: Gemini C18 (150 mm×4.6 mm, 5 um); Mobile Phase: A:0.01 M Ammonium Acetate (aq), B: Acetonitrile; Gradient: Time/% B: 0/10,1/10, 6/90, 8/98, 12/98, 12.01/10; Flow Rate: 1.0 mL/min; Diluent: Water

Method 7: Column: Xbridge C18 (100 mm×4.6 mm, 3.5 um); Mobile Phase: A:Acetonitrile, B: 10 mM Ammonium Bicarbonate in Aq; Gradient: Time/% B:0/95, 2/95, 4/5, 7/5, 8/95, 10/95; Flow Rate: 1.0 mL/min; Diluent:Acetonitrile:Water (1:1)

Method 8: Column: Aquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 um); MobilePhase: A: 0.1% Formic Acid in Acetonitrile, B: 0.1% Formic Acid inWater; Gradient: Time/% B: 0/97, 0.3/97, 3.2/2, 4/2, 4.01/97; ColumnTemp: 35° C.; Flow Rate: 0.6 mL/min

Method 9: Column: Aquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 um); MobilePhase: A: 0.1% Formic Acid in Acetonitrile, B: 0.1% Formic Acid inWater; Gradient: Time/% B: 0/97, 0.3/97, 5.5/2, 6/2, 6.01/97; ColumnTemp: 35° C.; Flow Rate: 0.6 mL/min

Method 10: Column: Aquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 um); MobilePhase: A: 0.05% Formic Acid in Water, B: 0.05% Formic Acid inAcetonitrile; Gradient: Time/% A: 0/97, 0.3/97, 3.2/2, 4.8/2, 5/97,5.10/97; Column Temp: 35° C.; Flow Rate: 0.6 mL/min

Method 11: Column: Aquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 um); MobilePhase: A: 0.05% Formic Acid in Water, B: 0.05% Formic Acid inAcetonitrile; Gradient: Time/% A: 0/97, 0.3/97, 3.5/2, 4.8/2, 5/97,5.01/97; Column Temp: 35° C.; Flow Rate: 0.6 mL/min

Method 12: Column: Aquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 um); MobilePhase: A: 0.05% Formic Acid in Water, B: 0.05% Formic Acid inAcetonitrile; Gradient: Time/% A: 0/95, 1.5/95, 3/85, 7/45, 10/5, 14/5,17/95, 20/95; Column Temp: 35° C.; Flow Rate: 0.6 mL/min

Reference Example 13-(4′-((trans)-2-((tert-butoxycarbonyl)((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)cyclopropyl)-[1,1′-iphenyl]-3yl)propanoicacid Step 1: Preparation of tert-butyl((trans)-4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)cyclohexyl)carbamate

Tert-butyl 4-oxocyclohexylcarbamate (22.1 g, 0.104 mol) was added to asolution of trans-2-(4-bromophenyl) cyclopropanamine (22 g, 0.104 mol)in DCE (400 mL) at 0° C., followed by the addition of AcOH (1.6 mL), andthe mixture was stirred at the same temperature for 5 min. Sodiumtriacetoxy borohydride (40 g, 0.187 mol) was slowly added portions wiseat 0° C. and the mixture was allowed to stir at RT for 16 h. Theprogress of the reaction was monitored by TLC. After completion, thereaction mixture was poured into saturated aq. NaHCO₃ solution (100 mL)and extracted with DCM (2×200 mL). The combined organic extracts werewashed with water (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄,filtered and filtrate was evaporated to get cis and trans mixture oftert-butyl(4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)cyclohexyl)carbamate (28g crude).

The crude compound was purified by flash column chromatography by using(SiO₂) EtOAc: pet ether (70:30), first the less polar tert-butyl((cis)-4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)cyclohexyl)carbamate(3 g) was isolated followed by the more polar tert-butyl((trans)-4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)cyclohexyl)carbamate(6 g, yield: 14.09%) obtained as a white solid, and also recovered 15 gof cis-trans mixture.

LC-MS (Method 1): Rt=4.87 min; m/z=409.3/411.4 (M+H⁺/M+2+H⁺).

Step 2: Preparation of tert-butyl((trans)-2-(4-bromophenyl)cyclopropyl)((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate

TEA (5.1 mL, 0.0367 mol) and (Boc)₂O (6.41 g, 0.0294 mol) were added toa solution of tert-butyl((trans)-4-(((trans)-2-(4-bromophenyl)cyclopropyl)amino)cyclohexyl)carbamate(Step 1 of Ref. example 1) (10 g, 0.0245 mol) in dry THF (100 mL) at 10°C. Then the reaction mixture was allowed to stir at RT for 16 h.Completion of the reaction was confirmed by TLC. The reaction mixturewas poured into water (200 mL) and extracted with EtOAc (2×100 mL). Thecombined organic extracts were washed with brine (100 mL), dried overanhydrous Na₂SO₄, filtered and filtrate was evaporated under reducedpressure. The crude was purified by silica gel column chromatographyusing EtOAc: pet ether (2:8) to afford tert-butyl((trans)-2-(4-bromophenyl)cyclopropyl)((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate(10.4 g, yield: 83.3%) as an off-white solid.

LC-MS (Method 2): Rt=3.23 min; m/z=531.15/533.19 (M+Na⁺/M+2+H⁺).

Step 3: Preparation of3-(4′-((trans)-2-((tert-butoxycarbonyl)((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)propanoicAcid

Pd (PPh₃)₄ (39.82 mg, 0.034 mmol) was added to a degassed solution oftert-butylpans)-2-(4-bromophenyl)cyclopropyl)((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate(Step-2 of Ref. example 1) (350 mg, 0.688 mmol), K₂CO₃ (285.2 mg, 2.066mmol) and 3-(2-carboxyethyl) phenyl boronic acid (0.826 mmol) inMeCN—H₂O (40 mL, 8:2) at RT. The mixture was again degassed for 5 minand heated to 90° C. for 16 h. Completion of the reaction was confirmedby TLC. The reaction mixture was diluted with EtOAc and filtered throughthe pad of Celite, the filtrate was concentrated and the crude compoundwas purified by preparative HPLC to afford3-(4′-((trans)-2-((tert-butoxycarbonyl)((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)propanoicacid (250 mg, yield: 62.8%) as an off-white solid

LC-MS (Method 4): Rt=4.63 min; m/z=577.0 (M−H⁺).

Reference Examples 2-4

The following compounds were obtained by following the proceduredescribed in reference example 1, but using suitable starting materials.

Ref LC-MS Rt Example Name Starting material method (min) m/z 23-(4′-((trans)2-((tert- trans-2-(4- 8 2.96 562.9butoxycarbonyl)(1-(tert- bromophenyl)cyclopropanamine (M − H⁺)butoxycarbonyl)piperidin-4- and tert-butyl 4-yl)amino)cyclopropyl)-[1,1′- oxopiperidine-1- biphenyl]-3-yl)propanoicacid carboxylate 3 3-(4′-((trans)-2-((tert- trans-2-(4- 8 3.01 576.8butoxycarbonyl)((1-(tert- bromophenyl)cyclopropanamine (M − H⁺)butoxycarbonyl)piperidin-4- and tert-butyl 4-yl)methyl)amino)cyclopropyl)- formylpiperidine-1- [1,1′-biphenyl]-3-carboxylate yl)propanoic acid 4 3-(4′-((1S,2R)-2-((tert- (1R,2S)-2-(4-10 3.30 601.4 butoxycarbonyl)((trans)-4- bromophenyl)cyclopropan- (M +Na⁺) ((tert- 1-amine andbutoxycarbonyl)amino)cyclohexyl)amino)cyclopropyl)- tert-butyl 4-[1,1′-biphenyl]-3-yl)propanoic oxocyclohexylcarbamate acid

The reference examples 1, 2, and 3 are a mixture of 2 isomers, whichcorrespond to the combination of the two different (trans) configurationregarding the cyclopropyl ring (which are (1R,2S) and (1S,2R),respectively) and the reference example 4 is a single (1S,2R) isomerregarding the cyclopropyl ring. The reference examples 1 and 4 contain acyclohexane ring with TRANS configuration.

Reference Example 5 (+)-Biotin-PEG₇-CH2CH2NH2.HCl(N-(23-amino-3,6,9,12,15,18,21-heptaoxatricosyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamidehydrochloride) Step 1: Preparation of (tert-butyl(25-oxo-29-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9,12,15,18,21-heptaoxa-24-azanonacosyl)carbamate)

HATU (0.97 g, 2.56 mmol) and DIPEA (0.67 mL, 3.84 mmol) were added to asolution of Boc-NH-PEG₇-CH₂CH₂NH₂ (tert-butyl23-amino-3,6,9,12,15,18,21-heptaoxatricosylcarbamate) (1.2 g, 2.56 mmol)and5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoicacid (0.7 g, 2.56 mmol) in DMF (12 mL). The reaction mixture was stirredat RT for 16 h. After completion, the reaction mixture was poured intoice water (50 mL) and extracted with 10% MeOH/DCM (2×40 mL). Thecombined organic extracts were washed with water (2×40 mL), brine (50mL), dried over anhydrous Na₂SO₄, filtered and filtrate was evaporatedto afford crude and it was purified by column chromatography (SiO₂) byeluting with 3% MeOH:DCM, to afford tert-butyl(25-oxo-29-((3aS,4S,6aR)-2-oxohexahydro-1-thieno[3,4-d]imidazol-4-yl)-3,6,9,12,15,18,21-heptaoxa-24-azanonacosyl)carbamate(600 mg, yield: 33.7%) as an off-white semi solid

LC-MS (Method 11): Rt=2.10 min; m/z=695.6 (M+H⁺)

Step 2: Preparation of (+)-Biotin-PEG₇-CH₂CH₂NH₂.HCl(N-(23-amino-3,6,9,12,15,18,21-heptaoxatricosyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4yl)pentanamidehydrochloride)

HCl in 1,4-dioxane (4M) (2.0 mL) was added to a solution of tert-butyl(25-oxo-29-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9,12,15,18,21-heptaoxa-24-azanonacosyl)carbamate(Step-1 of Ref. example 5) (400 mg, 0.57 mmol) in 1,4-dioxane (1.0 mL)at 10° C. The reaction mixture was allowed to stir at RT for 2 h. Aftercompletion, the reaction mixture was evaporated and the residue wastriturated with diethyl ether to afford (+)-Biotin-PEG₇-CH₂CH₂NH₂.HCl(N-(23-amino-3,6,9,12,15,18,21-heptaoxatricosyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamidehydrochloride) (300 mg, 83.4%) as a light brown gummy liquid.

LC-MS (Method 12): Rt=3.64 min; m/z=596.1 (M+H⁺)

Reference Example 6 (+)-Biotin-PEG₁₅-CH₂CH₂NH₂.HCl(N-(47-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45-pentadecaoxaheptatetracontyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamidehydrochloride)

This compound was obtained by following the procedure described inreference example 5, but using Boc-NH-PEG₁₅-CH₂CH₂NH2 instead ofBoc-NH-PEG₇-CH₂CH₂NH₂.

LC-MS (Method 3): Rt=3.59 min; m/z=947.6 (M+H⁺)

Reference Example 7 tert-butyl((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)((trans)-2-(3′-(3,41-dioxo-45-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-7,10,13,16,19,22,25,28,31,34,37-undecaoxa-4,40-diazapentatetracontyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)carbamate

A solution of (+)-Biotin-PEG₁₁-CH₂CH₂NH₂ (212.8 mg, 0.276 mmol) in DMF(0.5 mL) was added to a solution of3-(4′-((trans)-2-((tert-butoxycarbonyl)((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)propanoicacid (Ref. Example 1) (160 mg, 0.276 mmol), HATU (104 mg, 0.276 mmol)and DIPEA (56 mg, 0.414 mmol) in DMF (2.5 mL) at RT. The reactionmixture was stirred at RT for 16 h. Completion of the reaction wasconfirmed by TLC. The reaction mixture was poured into ice water (50 mL)and extracted with ethyl acetate (2×25 mL). The combined organic layerwas washed with saturated aq. NaHCO₃ solution (20 mL), brine (20 mL),dried over anhydrous Na₂SO₄, filtered and the filtrate was concentratedunder reduced pressure. The crude product was purified by preparativeHPLC to afford tert-butyl((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)((trans)-2-(3′-(3,41-dioxo-45-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-7,10,13,16,19,22,25,28,31,34,37-undecaoxa-4,40-diazapentatetracontyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)carbamate(50 mg, yield: 13.6%) as an off-white gummy solid.

LC-MS (Method 11): Rt=3.17 min; m/z=1331.9 (M+H⁺).

Reference Examples 8.12

The following compounds were obtained by following the proceduredescribed in reference example 8, but using suitable starting materials.

Ref LC-MS Rt m/z Example Name Starting material method (min) (M + H⁺) 8tert-butyl ((trans)-4-((tert- Ref. Example 1 11 3.19 1155.8butoxycarbonyl)amino)cyclohexyl)((trans)- and Ref. example 52-(3′-(3,29-dioxo-33- ((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)- 7,10,13,16,19,22,25-heptaoxa-4,28-diazatritriacontyl)-[1,1′- biphenyl]-4- yl)cyclopropyl)carbamate 9tert-butyl ((trans)-4-((tert- Ref. Example 1 12 4.82 1508.0butoxycarbonyl)amino)cyclohexyl)((trans)- and Ref. example 62-(3′-(3,53-dioxo-57- ((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49-pentadecaoxa- 4,52-diazaheptapentacontyl)-[1,1′-biphenyl]-4- yl)cyclopropyl)carbamate 10 tert-butyl 4-((tert- Ref.Example 2 9 3.89 1317.5 butoxycarbonyl)((trans)-2-(3′- and (+)-Biotin-(3,41-dioxo-45-((3aS,4S,6aR)-2- PEG₁₁- oxohexahydro-1H-thieno[3,4-CH₂CH₂NH₂ d]imidazol-4-yl)- 7,10,13,16,19,22,25,28,31,34,37-undecaoxa-4,40- diazapentatetracontyl)-[1,1′- biphenyl]-4-yl)cyclopropyl)amino)piperidine- 1-carboxylate 11 tert-butyl 4-(((tert-Ref. Example 3 8 2.58 1332.6 butoxycarbonyl)((trans)-2-(3′- and(+)-Biotin- (3,41-dioxo-45-((3aS,4S,6aR)-2- PEG₁₁-oxohexahydro-1H-thieno[3,4- CH₂CH₂NH₂ d]imidazol-4-yl)-7,10,13,16,19,22,25,28,31,34,37- undecaoxa-4,40-diazapentatetracontyl)-[1,1′- biphenyl]-4-yl)cyclopropyl)amino)methyl)piperidine- 1-carboxylate 12 tert-butyl((trans)-4-((tert- Ref. Example 4 10 2.97 1332.2butoxycarbonyl)amino)cyclohexyl)((1R, and (+)-Biotin-2S)-2-(3′-(3,41-dioxo-45- PEG₁₁- ((3aS,4S,6aR)-2-oxohexahydro- CH₂CH₂NH₂1H-thieno[3,4-d]imidazol-4-yl)- 7,10,13,16,19,22,25,28,31,34,37-undecaoxa-4,40- diazapentatetracontyl)-[1,1′- biphenyl]-4-yl)cyclopropyl)carbamate

The reference examples 8, 9, 10, and 11 are a mixture of 2 isomers,which correspond to the combination of the two different (trans)configurations regarding the cyclopropyl ring (which are (1R,2S) and(1S,2R), respectively) and the reference example 12 is a single (1S,2R)isomer regarding the cyclopropyl ring. The reference examples 8, 9 and12 contain a cyclohexane ring with TRANS configuration.

Example 1.1N-(39-(4′-((trans)-2-(((trans)-4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3yl)-37-oxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azanonatriacontyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamidedihydrochloride

HCl in 1,4-dioxane (4M) (0.5 mL) was added to a solution of tert-butyl((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)((trans)-2-(3′-(3,41-dioxo-45-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-7,10,13,16,19,22,25,28,31,34,37-undecaoxa-4,40-diazapentatetracontyl)-[1,1′-biphenyl]-4-yl)cyclopropyl)carbamate(Ref. Example 7) (50 mg, 0.037 mmol) in 1, 4-dioxane (0.3 mL) at 10° C.and the reaction mixture was stirred at 10° C. for 2 h. Completion ofthe reaction was confirmed by LCMS. The reaction mixture wasconcentrated under reduced pressure below 30° C., the residue wastriturated with EtOAc/diethyl ether and the obtained compound wasdissolved in deionized water and lyophilized to affordN-(39-(4′-((trans)-2-(((trans)-4-aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)-37-oxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azanonatriacontyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamidedihydrochloride (40 mg, yield: 89.8%) as an off-white solid

LC-MS (Method 5): Rt=3.82 min; m/z=1132.8 (M+H⁺).

Example 1.2-1.6

The following compounds were obtained by following the proceduredescribed in example 1.1, but using suitable starting materials.

LC-MS Rt m/z Example Name Starting material method (min) (M + H⁺) 1.2N-(27-(4′-((trans)-2-(((trans)-4- Ref. Example 8 11 1.81 955.7aminocyclohexyl)amino)cyclopropyl)- [1,1′-biphenyl]-3-yl)-25-oxo-3,6,9,12,15,18,21-heptaoxa-24- azaheptacosyl)-5-((3aS,4S,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4- yl)pentanamidedihydrochloride 1.3 N-(51-(4′-((trans)-2-(((trans)-4- Ref. Example 9 111.42 1307.7 aminocyclohexyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)-49-oxo- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45-pentadecaoxa-48- azahenpentacontyl)-5-((3aS,4S,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4- yl)pentanamidedihydrochloride 1.4 N-(37-oxo-39-(4′-((trans)-2- Ref. Example 10 6 5.501117.8 (piperidin-4- ylamino)cyclopropyl)-[1,1′- biphenyl]-3-yl)-3,6,9,12,15,18,21,24,27,30,33- undecaoxa-36- azanonatriacontyl)-5-((3aS,4S,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4- yl)pentanamidedihydrochloride 1.5 N-(37-oxo-39-(4′-((trans)-2- Ref. Example 11 6 5.571131.8 ((piperidin-4- ylmethyl)amino)cyclopropyl)-[1,1′-biphenyl]-3-yl)- 3,6,9,12,15,18,21,24,27,30,33- undecaoxa-36-azanonatriacontyl)-5- ((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4- yl)pentanamide dihdrochloride 1.6N-(39-(4′-((1S,2R)-2-(((trans)-4- Ref. Example 12 7 4.58 1132.0aminocyclohexyl)amino)cyclopropyl)- [1,1′-biphenyl]-3-yl)-37-oxo-3,6,9,12,15,18,21,24,27,30,33- undecaoxa-36- azanonatriacontyl)-5-((3aS,4S,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4- yl)pentanamidedihydrochloride

The chemical structures of the compounds of examples 1.1 to 1.6 areshown in the table below:

Example # STRUCTURE Example 1.1

Example 1.2

Example 1.3

Example 1.4

Example 1.5

Example 1.6

The examples 1.1, 1.2, 1.3, 1.4 and 1.5 are a mixture of 2 isomers,which correspond to the combination of the two different (trans)configurations regarding the cyclopropyl ring (which are (1R,2S) and(1S,2R), respectively) and the example 1.6 is a single (1S,2R) isomerregarding the cyclopropyl ring. The reference examples 1.1, 1.2, 1.3 and1.6 contain a cyclohexane ring with TRANS configuration.

The corresponding single (1S,2R) isomers of examples 1.2, 1.3, 1.4 and1.5 can be also prepared:

Other chemoprobes that can be prepared:

These chemoprobes can be prepared as a mixture of 2 isomers, whichcorrespond to the combination of the two different (trans)configurations regarding the cyclopropyl ring (which are (1R,2S) and(1S,2R), respectively) or as single (1S,2R) isomers regarding thecyclopropyl ring.

Biological Assays Example 2 Chemoprobe Inhibition of KDM1A Activity

The ability of the chemoprobes to inhibit KDM1A was tested in vitrousing the assay described below.

KDM1A inhibition assay: a multistep enzymatic reaction in which theenzyme first produces H₂O₂ during the demethylation of lysine 4 in a 21AA H3K4me2 N-terminal peptide. KDM1A chemoprobes were pre-incubated for15 min with human recombinant KDM1A enzyme (BPS Bioscience, Ref. 50100)on ice in the assay buffer (50 mM sodium phosphate pH 7.4). Theenzymatic reaction was initiated by the addition of K_(M) dimethylH3K4peptide substrate (Anaspec, Ref. 63677). After 30 min of incubation at37° C. Amplex Red reagent and the horseradish peroxidase (HRP) solutionwere added according to the recommendations of the supplier (Invitrogen)and left to incubate for 5 min at room temperature in the dark.Conversion of the Amplex Red reagent to resorufin, was monitored byfluorescence (λex=540 nm, λem=590 nm) using a microplate reader(Infinite F200 Tecan). Signals were corrected for background and theIC₅₀ value was calculated with GraphPad Prism Software.

In Table 1 we summarize the KDM1A IC₅₀ value of the all compoundsdescribed in example 1.

Example # KDM1A IC₅₀ (uM) 1.1 0.133 1.2 0.21 1.3 0.213 1.4 0.126 1.50.041 1.6 0.120

Example 3 Chemoprobe Mediated Precipitation of KDM1A

Preparation of samples and assay conditions:

Selected KDM1A chemoprobes (example 1.1 and 1.6) that have biotin tagincorporated were incubated with human recombinant KDM1A (Active Motif;#31334) or cell extracts obtained from KDM1A expressing THP-1 orMV(4;11) (human AML cell lines from ATCC) at increasing concentrationsof the chemoprobe. The capture agent used was streptavidin-coatedparamagnetic beads (ThermoFisher Scientific; #11205D). Cells were lysedin 1× Cell Lysis Buffer (Cell Signaling; #9803) containing 1× CompleteMini, Protease Inhibitor Cocktail Tablets (ROCHE; #11836153001) andchemoprobe (at the indicated concentrations, e.g. 5 μM for pull downassays) was added to frozen cell pellets containing 1·10⁶ cells. Tubeswere incubated for 5 min on ice and briefly sonicated to achieve lysis.The resulting extracts were centrifuged 10 min at 14.000×g at 4° C.Supernatants were collected and quantified by Bradford assay (BIO-RAD;#500-0006) following manufacturer's instructions.

250 μL of Dynabeads M-280 Streptavidin (ThermoFisher Scientific;#11205D) were washed 3 times and resuspended in 250 μL PBS. 350 μg ofprotein extracts (containing the chemoprobe) was then added andincubated for 30 min at room temperature (RT) using gentle rotation. Thechemoprobe-KDM1A complex bound to the streptavidin beads was separatedusing a magnet and washed 4 times in PBS. After the last wash, sampleswere resuspended in 25 μL of 3×SDS Sample Buffer and analyzed by westernblot. Samples were separated on a 10% Bis-Tris NuPAGE® Novex® Precastgel (Invitrogen; #P0301BOX) following manufacturer's instructions. Gelswere transferred onto nitrocellulose membrane (GE Healthcare; #RPN303D)for immunodetection by semi-dry transfer. N-specific binding sites wereblocked by incubating membranes in blocking buffer (5% non-fat dry milkin PBS-Tween 0.1%) at RT for 1 h. Nitrocellulose membrane was thenincubated in blocking buffer with an anti-KDM1A antibody (CellSignaling; #2184) diluted 1:1000 overnight (O/N) at 4° C. as a primarydetection reagent. After 5 washes in PBS-Tween 0.5%, the membrane wasincubated with a peroxidase-conjugated secondary antibody (JacksonImmunoresearch; #711-035-152) diluted 1:5000 for 1 h at RT. Signal wasdetected by enhanced chemiluminescence (ECL, Amersham, GE Healthcare;#W9643350) using a GeneGnome HR System (Syngene).

Results:

Titration analysis was performed using chemoprobe (at concentrations0.2-500 nM) to determine the probe concentration required to pull downthe protein complexes. The results shown in FIG. 1A demonstrate that thechemoprobe example 1.6 efficiently binds to KDM1A from THP-1 cells in adose-dependent manner and that its biotin group is readily accessible tothe streptavidin on the surface of the paramagnetic beads.

Four days of pretreatment of cells with increasing concentrations ofORY-1001 (0.2, 1, 5 and 25 nM) provoked a dose dependent reduction ofthe amount of KDM1A that could be pulled down using the chemoprobe 1.1or 1.6. Cellular extracts were obtained in presence of excess (5 uM)chemoprobe and proteins pulled down using magnetic streptavidinDynabeads analyzed by Western blot using KDM1A antibody. Pretreatmentwith 25 nM of ORY-1001 completely prevented pull-down of KDM1A withchemoprobe corresponding as example 1.1, indicating that KDM1Ainhibition was complete at this dose. The FIG. 1B shows the effect ofpretreatment of cells with different doses of ORY-1001 (nM) onchemoprobe mediated pull-down from THP-1 cells.

We also verified that other chemoprobes, such as Examples 1.4 and 1.5 at5 uM efficiently pulled down KDM1A from 500 ug of MV(4;11) proteinextracts. Pre-treatment of the cells with 25 nM of ORY-1001 completelyprevented pull-down of KDM1A with these chemoprobes.

Example 4 Chemoprobe Binding and Specificity Example 4.1 ChemoprobeSpecificity

Preferentially, the chemoprobes used for this invention selectively bindthe KDM1A target and do not bind other proteins, and more specifically,the chemoprobes do not bind the FAD cofactor in other structurallyrelated monoamine oxidases. The target selectivity of the chemoprobesfor binding FAD in KDM1A relative to other FAD containing enzymes isevaluated by testing the chemoprobes in different biochemical assays todetermine its MAO-A, MAO-B, IL4I1 and SMOX inhibitory activity in vitro.All IC50 values were calculated using Graph Prism Software.

Example 4.1.1 MAO Inhibition

Human recombinant MAO-A and MAO-B were purchased from Sigma Aldrich(#M7316 and #M7441 respectively). Enzymatic activities and inhibitionrate were analyzed in a fluorescence based assay using kynuramine (SigmaAldrich #K3250) as a substrate. Clorgiline and Deprenyl (Sigma Aldrich#M3778 and #M003) were used as controls for specific inhibition of MAO-Aand MAO-B respectively. Compounds were pre-incubated with protein for 15min on ice in 100 mM Hepes pH 7.5. The enzymatic reaction was initiatedby the addition of specific K_(M) Kynuramine and incubated 1 h at 37° C.The reaction was stopped by adding NaOH 2N (v/v). The conversion ofkynuramine to 4-hydroxyquinoline was monitored by fluorescence (λex=320nm, λem=360 nm) using a microplate reader. Chemoprobe (example 1.6) didnot inhibit MAO-A or MAO-B activity in the tested dose range, its MAO-Aand MAO-B IC50 is >100 uM.

Example 4.1.2 IL-411 Inhibition

Human recombinant IL-411 was purchased from RD Systems (#5684-A0-020).The biochemical activity was measured by its ability to oxidizephenylalanine and produce H2O2 in a horseradish-peroxidase coupled assayusing Amplex red as substrate. The assay buffer was 50 mM SodiumPhosphate pH 7; inhibition of IL-4I1 by compounds tested was monitoredduring 30 minutes in the presence of K_(M) phenylalanine. Chemoprobeexample 1.6 showed 31% of hIL-4I1 inhibition at 100 uM; its IC50was >100 uM.

Example 4.1.3 SMOX Inhibition

Mouse recombinant SMOX enzyme was provided by Professor Paolo Mariottini(Rome University). The biochemical activity was measured by its abilityto oxidize spermine in a horseradish peroxidase coupled assay usingAmplex red as a substrate. The assay buffer for mSMOX reaction was 100mM Sodium Phosphate pH 8.0. The inhibition by tested compounds wasmonitored during 10 minutes in the presence of K_(M) spermine. The mSMOXIC50 value of chemoprobe example 1.6 was 13 uM.

Example 4.1.4 Cellular Binding

To assess whether the chemoprobe binds selectively to KDM1A in cellularextracts, the KDM1A chemoprobe can be used to chemoprecipitate proteinsfrom cells. Chemoprobe example 1.6 inhibited recombinant SMOX with anIC50 of 13 uM. Chemoprobe example 1.6 was used to pull down targetprotein from MV(4;11) cells using magnetic streptavidin beads as acapture agent and analysis by western blot were performed as describedin example 3 using anti-SMOX antibody (Proteintec Europe; #15052-1-AP)diluted 1:200 or anti-KDM1A antibody as a primary detection reagentdescribed. As shown in FIG. 2, KDM1A was quantitatively recuperated bythe chemoprecipitation but SMOX was not detected, illustrating theselectivity of the probe for the KDM1A enzyme in cell extracts.

Example 5 Development of Chemoprobe Immunosorbent Assays to Detect Totaland Free KDM1A

We developed two independent KDM1A ELISA assays to determine both totaland free KDM1A in recombinant KDM1A full length protein or in proteinextracts from biological samples (e.g. cells or tissues) in a solidphase assay. In particular, to determine free KDM1A levels, a KDM1Achemoprobe-based immunosorbent assay was used. The assessment of thelevel of total and particularly free KDM1A is useful to evaluate thedegree of occupation of KDM1A (target engagement) by a KDM1A inhibitor,as described in more detail in Example 7.

Example 5.1 Selection of Specific KDM1A Antibodies Useful for ELISAApplications

To select a pair of antibodies with ability to bind KDM1A protein foruse in our assays, the following parameters were evaluated: theanti-KDM1A antibodies could recognize non-overlapping epitopes that didnot interfere with the streptavidin or other tag coupled to chemoprobenor with the interaction between KDM1A and Co-REST. Commercial antibodylists were scanned for antibodies that were preferentially monoclonaland announced to be functional in IHC, IP and WB. Preliminary modelingof the antibodies' epitope recognition sites on the KDM1A protein wasperformed in order to evaluate the diversity and the likeliness ofunwanted interactions, and candidate antibodies were selected forfurther functional evaluation. Said candidate antibodies were thenverified for their capacity to immunoprecipitate KDM1A protein fromfresh extracts from human cells. Two antibodies from different specieswere selected, mouse C-terminal mAb-KDM1A (Abcam #ab53269) hereindesignated as mAb-844, and rabbit N-terminal mAb-KDM1A (cell Signaling,#2184) herein designated as mAb-825, which can bind to KDM1A withoutinterfering with each other binding and without affecting the binding ofthe KDM1A chemoprobe example 1.6. Vice versa, inhibition of KDM1A withKDM1A inhibitors (and also with the chemoprobe) does not interfere withthe KDM1A recognition by the antibodies, and no steric hindrance wasdetected between antibody-antibody or antibody-probe and the capabilityto measure total KDM1A protein using cellular extracts.

mAb-825:

Rabbit N-terminal mAb-KDM1A (cell Signaling, #2184) that targets anepitope, designated herein EP1, located in the N terminal regionproximate to proline 60 (P60) of the human KDM1A sequence (UNIPROT ID060341-1) and blocked by peptide #LSD1 Blocking Peptide-2184 specific(Cell Signaling).

mAb-844:

mouse C-terminal mAb-KDM1A (Abcam #ab53269). This antibody targets anepitope, herein designated EP2, located on the C-terminal region andwhich comprises AMYTLPRQATPGVPAQ, corresponding to AA 832-847 of humanKDM1A.

Both antibodies recognize at least human, rat and mouse KDM1A protein.

Example 5.2 Luminescent KDM1A ELISA

For quantification of total and free amount of KDM1A protein in tissueand cell samples luminiscent KDM1A sandwich ELISA and KDM1A chemoprobeELISA assays were established. To measure total KDM1A levels, a sandwichELISA was employed using a surface-bound first anti-KDM1A antibody as acapture antibody (e.g. mAb-844) followed by a second antiKDM1A antibody(e.g. mAb-825) for detection. A horseradish peroxidase (HRP)-conjugatedsecondary antibody to the antiKDM1A antibody (mAb-825) was employed tomeasure total KDM1A bound to the plate surface. A schematicrepresentation of this sandwich ELISA assay to detect total KDM1A isdepicted in FIG. 3A. To measure free KDM1A levels, a chemoprobe-linkedimmunosorbent assay was used that employs streptavidin-coated plates tocapture a biotin tagged KDM1A chemoprobe bound to KDM1A and that uses anantiKDM1A antibody (e.g. mAb-825) as the detection antibody, using aHRP-conjugated secondary antibody as described before. A schematicrepresentation of this KDM1A chemoprobe-based ELISA assay to detect freeKDM1A is depicted in FIG. 3B.

Isolation of PBMCs from peripheral blood by density gradientcentrifugation:

Peripheral blood mononuclear cells (PBMCs) from rat were isolated fromperipheral fresh blood collected in anticoagulant tubes for blood (K2-or K3-EDTA as the preferred anticoagulant tubes) and diluted with anequal volume of PBS, using Ficoll-Paque PLUS reagent (GE Healthcare;#17-1440-02) or Leucosep Tubes (Greiner bio-one; #227288) according tomanufacturer's instructions. Cells were stained with tryptan blue andcell number and viability were determined using a Neubauer Chamber in anoptical microscope. Pellets of 1·10⁶ PBMCs were preserved at −80° C. Thesame procedure can be used for isolation of PBMCs from other resourcessuch as human or mouse blood. In the case of human samples, preferablyLeucosep procedure is used, whereas for mouse PBMCs, Ficoll procedure ispreferred.

Sample Preparation: Cells were lysed in presence of 25 nM KDM1Achemoprobe example 1.6 added to frozen cell pellets containing 1·10⁶cells following the conditions described in example 3. The same proteinextracts is used in sandwich ELISA developed for determination of totalKDM1A and in KDM1A chemoprobe capture ELISA developed for determinationof free KDM1A.

ELISA:

To quantify total KDM1A, the surface of Luminunc Plates Maxisorp (NUNC;#436110) were coated with a monoclonal anti-KDM1A antibody (Abcam;#ab53269, referred to as mAb-844) at 2 μg/mL in PBS as a capture agent.For quantification of free KDM1A, the surface of plates were coated withstreptavidin (Promega Biotech Ibérica; #Z7041) at 10 μg/mL in PBS as acapture agent. Coating was performed at 4° C. O/N. Wells were washed 3times in PBS, 0.1% Tween-20 and blocked with PBS-BSA 1% (Sigma; #A3059)for 2 h.

A KDM1A calibration curve of full length rKDM1A (Active Motif; #31334)diluted in PBS was included in each plate. Plates were then incubated 1h at RT and washed 5 times. Afterwards, monoclonal anti-KDM1A antibody(Cell Signaling; #2184, referred to as mAb-825), used as a primarydetection agent was diluted at 0.125 μg/mL in PBS and plates wereincubated 1 h at RT. After 6 washes, a secondary detection agent;peroxidase-conjugated secondary antibody (Jackson Inmunoresearch;#711-035-152) diluted 1:5000 was added to plates, incubated for 1 h atRT and plates were washed again. 100 μL/well of chemiluminescentsubstrate specific for horseradish peroxidase [HRP] in ELISA procedure(superSignal ELISA Femto Substrate, Invitrogen; #37074) was added.Plates were centrifuged during 30 sec to 1,500×g, shaken for 1 min at100 rpm and incubated inside the micropate reader (Infinite 200, Tecan)for 3 min at 25° C. The level of total KDM1A were determined usingrelative luminescence units (RLU) readouts that were acquired using a1000 ms integration time and 150 ms settle time. FIG. 3 represents theKDM1A ELISA assays developed, sandwich ELISA for determination of totalKDM1A (FIG. 3A) and KDM1A chemoprobe capture ELISA for determination offree KDM1A (FIG. 3B). The two determinations are run in two separateassays, HRP coupled secondary antibody directed against the anti-KDM1Aantibody reacts with the appropriate substrate to generate a luminescentsignal.

Results:

A dilution series of full length active recombinant rKDM1A was used toestablish a standard curve and to assess the dynamic range andsensitivity of the luminescent assays for total and free KDM1A. FIG. 4shows the standard curve of total and free human rKDM1A (Active MotifRef 31334, batch #3303004), both the sandwich ELISA and the KDM1Achemoprobe capture ELISA are conducted in presence of chemoprobe example1.6 (25 nM). As shown in FIG. 4, total and free KDM1A standard curvesexhibited linear behavior (r²>0.99). The ELISA LLOD (Lower Limit ofDetection) is around 10 pg of KDM1A and the detection range 15-2500 pg.

This curve can be used directly to determine the concentration (amount)of total KDM1A and free KDM1A in a biological sample if the efficiencyof detection of total and free recombinant KDM1A and of cellular totaland free KDM1A and the specific activity of the recombinant and cellularKDM1A is the same, or after applying the appropriate correction functionif efficiency of detection and specific activity of recombinant andcellular free KDM1A differ. The standard curve can be included in eachELISA assay as a positive control.

The above described KDM1A sandwich ELISA and KDM1A chemoprobe ELISAassays (both for total and free KDM1A), using the conditions andreagents described, can be used at least for the analysis of biologicalsamples from different species, including human, rat and mouse. They canalso be adapted to analyze samples from other animal species byselecting a suitable antibody for said species. Importantly, the methodcan be used to assess total and free KDM1A in clinically useful samples,p.e. in PBMCs obtained from human blood, using a PBMCs isolationprotocol, for example one of the PBMC isolation protocols disclosedabove. FIG. 5 shows the determination of the levels of total (FIG. 5A)and free (FIG. 5B) KDM1A in 7.5 ug of protein extract from PBMCs ofthree healthy donors (run in parallel with standard curve of rKDM1A oftotal and free KDM1A as a positive control). The data in FIGS. 5A and 5Bshow that free and total KMD1A levels can be determined in human PBMCs.

Example 5.3 Colorimetric KDM1A ELISA

The assay performed is similar to the assay disclosed above for theluminescent ELISA, but using Maxisorp plates (NUNC; #735-0013) as asurface and Tetramethylbenzidine (TMB) Liquid Substrate system(SIGMA-ALDRICH; #T0440) as substrate for the secondary detection agent.100 μLTMB was added per well and plates were incubated for 10 min beforestopping the reaction by addition of 100 uL/well of phosphoric acidsolution 2N. Absorbance was read at 450 nm using a microplate reader(Infinite 200, Tecan).

The detection range for this method, while being not as sensitive asluminescent ELISA, is still good and suitable for use in analysis ofbiological samples. Total and free KDM1A standard curves exhibitedlinear behavior (r2>0.99). The LLOD of colorimetric ELISA is around 200pg of KDM1A and the detection range (0.25-2.5 ng). As expected, superiorsensitivity was found when the ELISAs were run using chemiluminescence.

Example 6 Amplified Luminescent Proximity Homogeneous Assay to DetectTotal and Free KDM1A

The Amplified Luminescent Proximity Homogeneous Assay (Alpha) technologyis a homogeneous immunoassay platform that is an alternative toclassical ELISA assays such as the one described in Example 5.2.

The AlphaLISA assay detects the proximity of a first detection agentcontaining 250 nM latex donor beads containing phthalocyanine thatrelease singlet oxygen after irradiation at 680 nM to a second detectionagent containing similar sized acceptor beads containing Europium thatemit a narrow peak of light at 615 nM after absorption of singletoxygen. The AlphaPlex assay allows detection of multiple analytes in thesame assay volume and works similarly, but two acceptor beads are used(second and third detection agents) containing respectively Terbium orSamarium and emit light at 545 nM or 645 nM after absorption. Effectivesinglet oxygen transfer occurs when donor and acceptor beads areseparated by a distance of less than 200 nm.

The anti-KDM1A antibodies mAb-825 and mAb-844 selected (see example 5.1)to bind in close proximity to each other and to the protruding KDM1Achemoprobe example 1.6 on the surface of KDM1A. Binding of thechemoprobe did not affect the binding of the antibodies, nor did bindingto the surface of mAb-844 interfere with binding of mAb-825 in theexperimental settings used. The AlphaLisa/AlphaPlex format for thechemoprobe based immune assay was developed to assess KDM1A targetengagement.

For example, anti-KDM1A antibody (mAb-844) was conjugated to AlphaLISAacceptor beads (Perkin Elmer #6772001) to generate a first detectionagent, and anti-KDM1A antibody (mAb-825) was conjugated to AlphaLISAdonor beads (Perkin Elmer #6762013) to generate a second detection agentthat together form a detection system that can be used to determinetotal KDM1A levels. The first detection reagent can also be used incombination with a third detection reagent, streptavidin-coatedAlphaLISA donor beads (Perkin Elmer #6760002), targeting the biotin tagbearing KDM1A chemoprobe example 1.6, to form a second detection systemthat can be used to detect free KDM1A levels. AlphaLISA assay andconjugation of antibodies to the donor and acceptor beads was performedaccording to the vendor's instructions.

To illustrate the use of AlphaLISA to detect total and free KDM1A, adilution series of human rKDM1A was prepared, incubated with 100 nMchemoprobe example 1.6 and mixed with anti-KDM1A mAb-844 conjugatedAlphaLISA acceptor beads and either anti-KDM1A mAb-825 conjugatedAlphaLISA donor beads or streptavidin coated AlphaLISA donor beads todetect free (FIG. 6A) or total (FIG. 6B) KDM1A respectively. Theresulting curves are bell-shaped and characterized by a titratablesignal decrease (hook effect) following a plateau obtained after aninitial concentration-dependent signal increase, a pattern typical forAlphaLISA assays. The maximum signal to background ratio was obtainedwith 100 nM rKDM1A, i.e. at equimolar concentrations of chemoprobe andenzyme. As expected, the free KDM1A signal was blocked by pretreatmentof the enzyme with ORY-1001 (5 uM), as the KDM1A inhibitor binds toKDM1A, so that there is no free KDM1A available for binding to the KDM1Achemoprobe; see FIG. 60, where rhombi corresponds to rKDM1A+KDM1Achemoprobe (Example 1.6, 100 nM), circles correspond to rKDM1A+KDM1Achemoprobe (Example 1.6, 100 nM)+ORY-1001 (5 uM) and trianglescorrespond to negative control (rKDM1A protein alone).

For the multiplex assay, the phthalocyanine containing Alphaplex donorbeads can be coupled to one of the anti-KDM1A antibodies (p.e. mAb-825)and Terbium containing Acceptor Beads to the second anti-KDM1A antibody(p.e mAb-844) and Samarium containing AlphaPLEX 645 StreptavidinAcceptor Beads can be used to bind the chemoprobe example 1.6 or viceversa, and detect total and free KDM1A simultaneously.

Example 7 Use of Chemoprobe for Assessment of KDM1A Target Engagement inCells

The chemoprobe-based immunoassay described above can be used to assessKDM1A target engagement in cells, as shown below.

7.1 Method:

To establish a dose curve, MV(4;11) cells were treated for 96 h withvehicle or increasing doses of ORY-1001. Cells were lysed and proteinextracts were obtained in the presence of 25 nM of chemoprobe (example1.6) as described in example 5.2. Total protein extracts were quantifiedand 1 ug of total protein was used to perform the target engagementanalysis as described above, using the luminescent substrate.

7.2 Target Engagement Calculation

After treatment of cells with a KDM1A inhibitor such as ORY-1001, a partof free KDM1A enzyme can be occupied by the compound. When the KDM1Afrom these cells is exposed to an excess of the KDM1A chemoprobe such asexample 1.6, it will rapidly bind to the FAD cofactor and tag the freeKDM1A. The chemoprobes and the method described herein can be used toassess direct KDM1A occupation (target engagement) of ORY-1001 or anyother KDM1A inhibitor in cells, tissues and/or other biological samplesthat express KDM1A.

To determine the target engagement (TE), a relative quantificationmethod was used, in which the target engagement in a given sample X wascalculated relative to a reference sample REF (pre-treatment or vehicle)of the same nature. After raw data processing (robust elimination ofoutliers in replica analysis points according to Grubbs criteria andsubtraction of blank signal), the target engagement was calculated asTE _(X)=1−(R _(X) /R _(REF)); where R _(X)=(RLU_(Free,X)/RLU_(Total,X))and R _(REF)=(RLU_(Free,REF)/RLU_(Total,REF));

or, expressed in %TE _(X)(%)=100−((R _(X)(%)/R _(REF)(%))×100); whereR×(%)=(RLU_(Free,X)/RLU_(Total,X))×100 and R_(REF)(%)=(RLU_(Free,REF)/RLU_(Total,REF))×100.

7.3 Results:

Total and free KDM1A levels, measured in RLUs, were determined followingthe conditions described in example 5.2, and target engagement wascalculated following the method described in 7.2 above. FIG. 7 shows the% of target engagement plotted against ORY-1001 concentration, and theIC50 value for target engagement of ORY-1001 to KDM1A in MV(4;11) wascalculated to be 0.35 nM using Graph Prism Software.

Using the same method, we have also evaluated KDM1A target engagement byother KDM1A inhibitors such as compound A and compound B after 24 h oftreatment in MV(4;11) cells. Compound A (or Comp A) is the compound withchemical name(trans)-2-Phenyl-N-(piperidin-4-ylmethyl)cyclopropan-1-amine, which isdisclosed in example 5 of WO2013/057320. Compound B (or Comp B) isparnate, also known as tranylcypromine, with chemical nametrans-2-Phenylcyclopropylamine. The results are shown in FIG. 8A, wherethe % of KDM1A target engagement for ORY-1001 (at 0.1, 1 and 10 nM),Comp A (at 0.1, 1 and 10 nM) and Comp B (at 10 nM, 1 mcM and 10 mcM) areplotted. FIG. 8A shows a dose-responsive cellular target engagement inaccordance with their biochemical KDM1A activity (IC50_(LSD1)=0.018 uM,0.022 uM and 15 uM for ORY-1001, comp A and B, respectively).

The methods and chemoprobes disclosed herein have also been used todetermine target engagement of KDM1A inhibitors in additional celllines, including other leukemic cell lines like THP-1 and solid tumorcell lines like prostate cancer and small cell lung cancer (SCLC) celllines. For example, leukemia THP-1 cells or prostate cancer LNCap cellswere treated for 24 h with ORY-1001 (at concentrations in the range0.25-25 nM in THP-1 cells, at 0.1, 1 and 10 nM in LNCap cells), thenlysed in Cell Lysis Buffer 1× (Cell Signaling Technology) in presence ofthe chemoprobe example 1.6 (25 nM) as described above, and free andtotal KDM1A levels were determined as described in Example 5.2 and usedto calculate target engagement as described above. The % of Targetengagement obtained in THP-1 cells treated with ORY-1001 is shown inFIG. 8B. The results show a dose-responsive cellular target engagement,with a calculated IC50 value using Graph Prism Software for targetengagement of ORY-1001 to KDM1A in THP-1 cells of 0.6 nM. The targetengagement data obtained in LNCap cells treated with ORY-1001 is shownin FIG. 8C, where a clear KDM1A target engagement of the KDM1A inhibitortested is seen.

The results provided in this Example 7 show that the chemoprobe-basedmethods of the invention can be used to reliably determine targetengagement of KDM1A inhibitors in different kinds of cells, includingleukemic and solid tumor cells.

Example 8 Use of Chemoprobe for Assessment of In Vivo KDM1A TargetEngagement Using Ex Vivo Samples

The chemoprobes and chemoprobe-based methods of the invention have beensuccessfully used to determine KDM1A target engagement in livingsubjects like animals (for example rat or mice) and humans that havebeen treated with a KDM1A inhibitor, using samples taken from saidsubjects (ex vivo samples).

For example, as shown in sections 8.1 to 8.3, rats or mice have beenadministered a KDM1A inhibitor at different doses or vehicle, andsamples have then been obtained from said animals (for example bloodsamples, or tissue or organ samples) and analyzed to determine free andtotal KDM1A levels in said samples, which have been used to determineKDM1A target engagement by the KDM1A inhibitor as described above.Likewise, using the same approach, KDM1A target engagement has also beenshown in humans treated with a KDM1A inhibitor, as described in moredetail in Example 9 below.

8.1. Samples preparation and obtention of protein extracts from cellswas performed as described in example 5. Frozen tissues were homogenizedwith a mortar and pestle chilled on dry ice. Powdered samples wereresuspended in lysis buffer and forced through a 18 gauge blunt syringeneedle to lyse the cells, proteins were then extracted as describedabove for cell pellets (example 5.2). Isolation of PBMCs from peripheralblood was also as described in example 5.2.

8.2. Assay Protocol and Calculation of TE: same as Described in Example7.1 and 7.2.

8.3. Results:

The luminescent ELISA method was used to assess KDM1A target engagementusing ex-vivo samples of rats treated with ORY-1001 by oral gavageduring 4 consecutive days. Four different doses were used (1, 3, 10 and30 ug/kg/day). The KDM1A target engagement was calculated as describedpreviously (see example 7), where R_(X) corresponds to each conditionanalyzed and R_(REF) is the sample corresponding to vehicle (nontreated) rat PBMCs. FIG. 9A summarizes the KDM1A target engagement (in%) in pooled rat PBMC (n=3) treated with ORY-1001 at each dose testedand isolated two hours after last administration, and FIG. 9B shows theKDM1A target engagement (in %) in pooled rat lung (n=3) from the sameanimals treated with ORY-1001. A clear dose-response of KDM1A targetengagement was observed to ORY-1001 treatment in both PBMCs and lung.

The methods of the invention have also been used to determine KDM1Atarget engagement in SAMP8 mice (a non-transgenic model forneurodegeneration reminiscent of Alzheimer's disease) which have beentreated with the KDM1A inhibitor designated as Compound C. Compound C isthe compound with chemical name(−)5-((((trans)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine,which is described in WO2012/013728. This compound inhibits KDM1A withan IC50 (biochemical assay) of 101 nM, is orally available and has beenshown to cross the blood-brain barrier. Female SAMP8 mice were treatedwith Compound C starting at 5 months of age and during 2 months. Micewere randomly distributed in three groups, receiving vehicle or CompoundC at 0.11 mg/kg/day or 0.32 mg/kg/day, respectively. Compound C wasdiluted in vehicle (1.8% hydroxypropyl-beta-cyclodextrin, Sigma-Aldrich)and administered in drinking water. The dose was calculated according tothe animal water consumption average per cage and adjusted weekly.Compound C (or vehicle) was available for 5 days followed by a 2 dayclearance in a weekly period. After 2 months of treatment, animals weresacrificed and brains were dissected and snap frozen on dry ice forfurther TE analysis, which was performed following the proceduredescribed previously. The results are shown in FIG. 9C, which shows themean % KDM1A target engagement for each dose of Comp C tested; a cleardose-response of KDM1A target engagement was observed to Compound Ctreatment in brain.

The data reported in this Example 8 confirm the usefulness of thechemoprobes and chemoprobe-based methods of the invention to monitorKDM1A target engagement in living organisms.

Example 9 Use of Chemoprobe for Analysis of Pharmacodynamics andAssessment of KDM1A Target Turn-Over

The chemoprobes and methods according to the invention have been used toquantify KDM1A target engagement and assess pharmacodynamics of bindingof KDM1A inhibitors to KDM1A in animals and humans. In this type ofstudy, typically animals (for example rats or mice) or humans (forexample in the course of a clinical trial either using healthyvolunteers or patients) are administered a KDM1A inhibitor, either as asingle dose or as repeated doses (over several days or months) andsamples are taken from said subjects at different time points, forexample before start of the treatment with the KDM1A inhibitor and atdifferent times after treatment has started, and target engagement isdetermined in said samples using the methods described above.

Rats:

Obtention of proteins extracts, assay protocol and calculation of TE asdescribed in example 8.1, 7.1 and 7.2 respectively.

To assess the dynamics of target engagement in vivo, vehicle or ORY-1001was administered p.o. to rats either 1 day or during 5 consecutive days,at a dose of 2.5 and 15 ug/kg/day. PBMCs were obtained pre-dose (beforestarting the drug administration) and 2, 6, 24 h after the single doseadministration, or pre-dose (i.e. 0 h) and 2, 6, 24, 48 and 72 h afterthe last drug administration in the multiple dose administration. Totaland free KDM1A protein levels in PBMCs were assessed using 0.5 ug oftotal protein extracts. FIG. 10 shows the percentage of KDM1A targetengagement of pooled rat PBMCs (n=3) treated with ORY-1001 (2.5 and 15ug/Kg/day) administered orally as a single dose (1) and as a multipledose (during 5 days, 11111). The KDM1A target engagement was calculatedas described previously (see section 7.2)), where R_(X) corresponds toeach time-point analyzed and R_(REF) is the sample corresponding to thepre-dose of rat PBMC. FIG. 10 shows a clear time- and dose-response inKDM1A target engagement. At the same dose level, target engagement aftermultiple administrations is higher than single administration. Also asshown in FIG. 10, after the last dose in absence of further treatment,the percentage of KDM1A target engagement gradually decreases.

Humans

A Phase I clinical study has been performed to characterize the safety,tolerability, pharmacokinetics and pharmacodynamics of Compound C inhealthy volunteers. The first stage of this clinical study is arandomized, double-blind, dose escalation study, in which the KDM1Ainhibitor was administered as a single oral dose in capsules. Up to fivedifferent dose levels were tested in cohorts of 8 volunteers, randomizedto receive either Compound C (n=6) or placebo (n=2). As part of thepharmacodynamic evaluations in this clinical study, KDM1A targetengagement (TE) was assessed at pre-treatment, and 12 and 72 h afterdrug administration. Briefly, blood samples (10 mL) were collected inEDTA-K2 tubes from all the treated volunteers (active drug and placebo,n=8 per dose level) and PBMCs pellets obtained by density gradientcentrifugation in Leucosep™ tubes and stored at −90° C. until analysis.Only PBMC samples meeting pre-established sample quality controlcriteria are used for the TE analysis. In particular, samples with signsof hemolysis and/or with traces of red cells are discarded.

KDM1A TE values obtained for each volunteer in the cohort of volunteerscorresponding to the single dose of 1.5 mg of Compound C are shown inFIG. 11. Clear KDM1A target engagement was observed at 12 and 72 hpost-administration in 6 volunteers, whereas KDM1A TE levels of 0% wasobserved in the remaining two subjects. These results show that thechemoprobes and methods of the invention are useful to determine KDM1Atarget engagement by KDM1A inhibitors in human clinical samples.

Example 10 Chemoprobe Mediated Chemoproteomics of KDM1A

The KDM1A chemoprobe can be used to chemoprecipitate KDM1A containingcomplexes and to identify KDM1A interacting protein partners by means ofWestern blot analysis, antibody microarray analysis or massspectrometry.

Selected chemoprobes (example 1.1 and 1.6) are incubated with cellextracts obtained from vehicle or KDM1A inhibitor (here used as anegative control) treated THP-1 or MV(4;11). Cells are lysed in 1× CellLysis Buffer (Cell Signaling; #9803) containing 1× Complete Mini,Protease Inhibitor Cocktail Tablets (ROCHE; #11836153001) and KDM1Achemoprobe example 1.6 (5 μM for pull down) is added to frozen cellpellets at a ratio of 200 μl Cell Lysis Buffer to 10-mg of cell pellet(10⁷ cells are used). Tubes are incubated for 5 min on ice and brieflysonicated to achieve lysis. The resulting extracts are centrifuged 10min at 14.000×g at 4° C. Supernatants are collected and quantified byBradford assay (BIO-RAD; #500-0006) following manufacturer'sinstructions. 250 μL of Dynabeads M-280 Streptavidin (ThermoFisherScientific; #11205D) are used as a capture agent and are washed 3 timesand resuspended in 250 μL PBS. Protein extracts (containing thechemoprobe) are then added and incubated for 60 min in ice using gentlerotation.

The chemoprobe-KDM1A complex bound to the streptavidin beads isseparated using a magnet and washed 4 times in PBS or Cell Lysis Buffer,prior to elution. The eluted proteins may then be analyzed usingdifferent techniques like western blot, antibody array analysis or massspectrometry, as described for example below.

Western Blot:

After the last wash, samples are resuspended in 25 μL of 3× denaturingSample Buffer Samples are separated on a 10% Bis-Tris NuPAGE® Novex®Precast gel (Invitrogen; #P0301BOX) following manufacturer'sinstructions. Gels are transferred onto nitrocellulose membrane (GEHealthcare; #RPN303D) for immunodetection by semi-dry transfer.N-specific binding sites are blocked by incubating membranes in blockingbuffer (5% non-fat dry milk in PBS-Tween 0.1%) at RT for 1 h.Nitrocellulose membrane are incubated in blocking buffer with ananti-KDM1A antibody (Cell Signaling; #2184) diluted 1:1000 over night(O/N) at 4° C. or with antibodies of interest as a primary detectionreagent; according to the vendor's instructions. After 5 washes inPBS-Tween 0.5%, the membrane is incubated with a peroxidase-conjugatedsecondary antibody (Jackson Inmunoresearch; #711-035-152) diluted 1:5000for 1 h at RT as a secondary detection reagent. Signal is detected byenhanced chemiluminescence (ECL, Amersham, GE Healthcare; #W9643350)using a GeneGnome HR System (Syngene).

Antibody Array Analysis

The chemoprobe precipitates obtained from the vehicle and the KDM1Ainhibitor treated cells are labeled according to the vendor'sinstructions using the Amersham Cy5 or Cy3 Monoreactive Dye Pack (GEHealthcare PA25001/PA23001), respectively. Antibody microarrayscontaining potential capture agents to the KDM1A interacting proteins inthe chemoprobe bound KDM1A complex are incubated overnight in 1×PBS/0.2%NP-40 and then incubated for 1 h at room temperature in blocking buffersodium tetraborate decahydrate (2.85 g/100 ml) ethanolamine (453 ul/100ml), in Agilent slide gaskets (1-well) and hybridization chambers;protected from light in an Agilent gasket rotator at speed 5. Afterthis, the gaskets are dismounted and 500 ul of Cy3/Cy5 labeled proteinis pipeted on the slide and mounted in the gasket for 1 h in the sameconditions as used for blocking. After this incubation, the slides openin wash buffer 3 and washed for 5 min in wash buffer 1 (2×PBS 0.5%NP-40), 2× in wash buffer 2 (1×PBS/0.5% NP-40); 2× in wash buffer 3(1×PBS/0.2% NP-40) and aqua milliQ (3×), after which they are dried andscanned in an Agilent scanner. Data acquisition is performed usingPC_Scan (Agilent Scan Software v.7.0 o sup.).

Mass Spectrometry:

Elution for mass spectrometry protein identification can be performedincubating the Dynabeads in 2% SDS for 45 minutes (in a heating block at95 C) or by incubation in 8 M Urea pH 8.5 for 30 minutes (at roomtemperature). Proteins are then treated with Dithiothreitol (DTT) andIodoacetamide (IAA) to break disulfite bonds, prior to Tricloroaceticacid (TCA)/aceton precipitation and overnight (16 hours approximatelty)digestion with 1 μg of porcine Tripsin. After stopping the reaction with1 μL of formic acid 1%, 1-5 μL are injected for separation in the HPLCAS2nanoULTRA (EKSIGENT) apparatus. Protein identification has beenperformed using a LQT-Velos-Orbitrap (Thermo Scientific) massspectrometer.

For example, a KDM1A chemoproteomic analysis using MS for proteinidentification was performed as follows:

To identify interacting proteins and transcription factors recruitingKDM1A to its target sites in the genome, pull-down experiments wereperformed in MV(4;11) cells with an active KDM1A-specific chemoprobe,using KDM1A inhibited cells or a mock pull down product as controls.Specifically, MV(4;11) cells were treated with either DMSO (0.1%) orwith an irreversible KDM1A inhibitor (200 nM). Pellets of 10⁷ cells wereprepared after 4 days of treatment and frozen at −80° C. For thepulldown, cells were resuspended in 400 μL of Cell Lysis Buffer (CellSignaling) supplemented with 100 nM KDM1A chemoprobe of example 1.6.Samples were maintained in ice for 5 minutes, prior to sonication andhigh speed centrifugation to separate cellular debris. Cleared lysateswere then incubated 1 hour on ice, to allow binding of the chemoprobe.For the pulldown, 150 μL of streptavidin beads (Life Technologies) wereadded to 450 μg of protein in 300 μL of Cell Lysis Buffer and incubatedfor 30 minutes at room temperature on a spinning wheel. After magneticisolation, the beads were washed 5 times with ice-cold PBS to removenon-specific binding. Elution was performed using 25 μL of 1% SDS,incubating the beads for 15 minutes at 95° C. Eluted protein wereprecipitated with Trichloroacetic Acid, resuspended in 8M urea pH 8.0and digested overnight with porcine trypsin (1 μg). Peptides wereidentified by LC-MS, using an AS2nanoULTRA HPLC system (Eksingent) forseparation and a LTQ-Velos-Orbitrap detector (Thermo Scientific).Protein lists were processed by String (http://string-db.org/)(76), forgenerating the final graphical representation of the protein networksassociated to KDM1A. Proteins enriched by chemoprobe pull-down inuntreated relative to treated MV(4;11) cells included, among others,components of the CoREST complex, in accordance with published reportsin the scientific literature that have described KDM1A to be associatedwith CoREST.

This confirms the KDM1A chemoprobes of the invention are useful toidentify KDM1A interacting protein partners.

Example 11 Chromatin Immuno Precipitation (ChIP) Assays

ChIP assays can be performed using for example the SimpleChiP EnzymaticChromatin IP Kit (Cell Signaling, 9003), according the suppliersindications. Briefly, THP-1 cells are treated with vehicle or 20 nMORY-1001 for 96h, fixed with 1% formaldehyde (v/v) for 10 min at roomtemperature. Fixation is stopped by addition of glycine. Cell are lyzedin presence of a KDM1A chemoprobe including a biotin tag (example 1.6)and chromatin digested with the micrococcal nuclease from S. aureus(Roche) for 20 min at 37° C. and sonicated with bath sonicator (CD-4820COBOS) 5×30s ON/OFF at high power to produce chromatin fragments of150-300 bp. Lysates are subjected to pull-down using magneticstreptavidin beads as a capture agent. After pull down of chromatincomplexes, the protein-DNA cross-links are reversed and DNA is purified.Pulled-down sequences are analyzed by next-generation sequencinganalysis (ChIP-Seq) or by qPCR using specific primers of a gene ofinterest.

Example 12 Chemohistochemistry

The KDM1A chemoprobe incorporating a label or tag, such as fluoresceinlabeled or biotin labeled probes, can be used for histologicallocalization of KDM1A in fresh or fixed tissues. If active KDM1A ispresent in the sample the probe will be able to join covalently to theenzyme habilitating direct localization by fluorescence if thefluorescein labeled probe is used and/or by secondary detection using anantibody against fluorescein or streptavidin (if the biotin labeledprobe is used) conjugated to a fluorophore. Alternative to fluorescence,enzymatic secondary detection could be used using horseradishperoxidase, alkaline phosphatase or any other enzymatic reaction with achromogenic product. The assay would allow locating active KDM1A in anycellular or tissue compartment. Combined with immune detection of KDM1Athe assay would be used to calculate the free/total KDM1A ratio in thetarget tissue.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains.

All publications, patents and patent applications cited herein arehereby incorporated herein by reference in their entireties.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the appendedclaims.

The invention claimed is:
 1. A compound of formula (II)

or a salt thereof, wherein: P is a tag or label; D is cyclyl or —(C₁₋₄alkylene)-cyclyl, wherein cyclyl and the cyclyl moiety in the —(C₁₋₄alkylene)-cyclyl are each optionally substituted; L₁ is a divalent C₆₋₉₀hydrocarbon group, wherein one or more carbon atoms comprised in saidhydrocarbon group are each optionally replaced by a heteroatom selectedindependently from O, S and N, wherein one or more carbon atomscomprised in said hydrocarbon group are each optionally replaced by agroup selected independently from the group consisting of —C(═O)—,—NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,—O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—, —SO₂—NR¹— and—NR¹—SO₂—, and wherein L₁ provides a distance of at least 3 atomsbetween P and X₁; R¹ is hydrogen or C₁₋₄ alkyl; X₁ is —C(═O)—, —NR¹—,—NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,—O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—, —SO₂—NR¹—, —NR¹—SO₂—or —O—; X₂ is C₀₋₅ alkylene; one of Z₁ and Z₂ is CH or N, and the otherof Z₁ and Z₂ is CH; s and t are each independently selected from 0, 1and 2; R₅ and R₆ are at each occurrence independently selected from C₁₋₄alkyl, halo, —NH₂, —NR^(a)R^(c), —CN, —OH, —OR^(c), haloC₁₋₄ alkyl,cyclyl, cyclylC₁₋₄ alkyl-, C₁₋₄ alkyl-O—C₁₋₄ alkyl; R^(a) is selectedfrom hydrogen, C₁₋₄ alkyl and haloC₁₋₄ alkyl; R^(c) is independentlyselected from C₁₋₄ alkyl, haloC₁₋₄ alkyl, cyclyl, cyclylC₁₋₄ alkyl-, andC₁₋₄ alkyl-O—C₁₋₄ alkyl; and wherein the phenyl and —NH-D groups on thecyclopropyl ring are in trans configuration.
 2. The compound of claim 1,has formula (IIa), or a salt thereof


3. The compound of claim 1 wherein D is cycloalkyl, benzocycloalkyl,heterocycloalkyl or —(C₁₋₄ alkylene)-cyclyl, wherein the cycloalkyl, thebenzocycloalkyl, the heterocycloalkyl and the cyclyl moiety in the—(C₁₋₄ alkylene)-cyclyl are each optionally substituted.
 4. The compoundof claim 1, which has formula (III), or a salt thereof

wherein the substituents on the cyclopropyl ring are in transconfiguration.
 5. The compound of claim 1 wherein X₁ is —NHC(═O)— or—C(═O)NH—.
 6. The compound of claim 1 wherein X₁-X₂ is a group offormula

wherein the group is linked to the ring through the carbon atom and toL₁ through the N atom.
 7. The compound of claim 1, wherein each of Z₁and Z₂ is CH.
 8. The compound of claim 1, wherein each of s and t is 0.9. The compound of claim 1, wherein L₁ is a group of formula X₄-X₅,wherein X₄ is linked to P and X₅ is linked to X₁, wherein: X₄ is—C(═O)—, —NR¹—, —NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—,—NR¹—C(═S)—NR¹—, —O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, —O—C(═O)—,—SO₂—NR¹— or —NR¹—SO₂—; and X₅ is a group of formula (i) or (ii): (i)—(CH₂CH₂O)_(n)—(CH₂)_(p)—, wherein n is an integer from 6 to 20, and pis an integer from 1 to 5; or (ii)—(CH₂CH₂O)_(x)(CH₂)_(q)-G-(CH₂CH₂)_(y)(CH₂)_(r)—, wherein G is—NR¹—C(═O)—, —C(═O)—NR¹—, —NR¹—C(═O)—NR¹—, —NR¹—C(═S)—NR¹—,—O—C(═O)—NR¹—, —NR¹—C(═O)—O—, —C(═O)—O—, or —O—C(═O)—, one of x and y isan integer from 1 to 18 and the other from x and y is an integer from 0to 17 provided that x+y is from 4 to 18, and each of q and r is aninteger independently selected from 1 to
 5. 10. The compound of claim 1,wherein P is biotin or a biotin derivative or P is a fluorescent label.11. The compound of claim 1 selected from

and salts thereof.
 12. The compound of claim 1, wherein the compound is

or a salt thereof.